CHIMERIC PROTEINS FOR TARGETING dsRNA

ABSTRACT

Described herein are recombinant chimeric proteins comprising a double stranded RNA (dsRNA) binding domain and a cancer-cell targeting domain for targeting of dsRNA to cancer cells. Methods of use of the described chimeric proteins are also provided herein.

FIELD OF THE INVENTION

The present invention relates in general to recombinant chimericproteins comprising a double stranded RNA (dsRNA) binding domain and acancer-cell targeting domain for targeting of dsRNA to cancer cells.

Provided herein are recombinant chimeric proteins comprising a doublestranded RNA (dsRNA) binding domain and a cancer-cell targeting domainfor targeting of dsRNA to cancer cells. Methods of use of the describedchimeric proteins are also provided herein.

BACKGROUND OF THE INVENTION

Selective delivery of drugs to tumor cells can improve efficacy andreduce toxicity. Selectivity can be obtained by utilizing a drug vehiclethat can distinguish between the targeted malignant cells and untargetednon-malignant cells. High specificity towards cancer can be programmedinto recombinant proteins by fusing targeting moieties and drug bindingmoieties. The targeting moiety must recognize cell surface moleculesthat are uniquely expressed on cancer cells but not on non-cancerouscells or are over-expressed in cancer cells as compared to their normalcounterparts. One appropriate target is the Epidermal Growth FactorReceptor (EGFR), which is over-expressed in multiple types of humancancer and is usually associated with aggressive disease and lowsurvival rate [25]. EGFR over-expression can be utilized to selectivelydeliver high quantities of polyinosine/polycytosine (polyIC) into tumorcells, while leaving normal cells unaffected, due to the low amounts ofpolyIC delivered. PolyIC is an attractive anti-tumor agent, as it caninduce cancer cell apoptosis by activating Toll-Like Receptor 3 (TLR3)in cancer cells [26-30]. Furthermore, TLR3 activation by polyIC triggersthe induction of cytokines, chemokines and other pro-inflammatorymediators [31-34], thus reinstating anti-tumor immunity [35, 36].However, the use of polyIC is limited by its extreme toxicity andinefficient cellular uptake when delivered systemically [37, 38].

In order to limit toxicity and increase cellular uptake we have beendeveloping vehicles for the targeted delivery of polyIC directly totumors. In our previous studies, we employed chemical vectors that bindpolyIC electrostatically, and utilize EGF or anti-HER2 affibody ashoming entities towards EGFR or HER2 [38-42].

Further, prostate cancer is the second most commonly diagnosed cancerworldwide, accounting for over 25% of new cancer cases diagnosedannually among men in the US (1). In the case of metastatic prostatecancer, patients are mostly treated with androgen deprivation therapy(ADT).

While this therapy generally achieves a short-term remission, patientstypically develop castration-resistant prostate cancer (CRPC). There isa great demand for novel therapies for CRPC patients, as these patientsrarely respond to existing therapies and demonstrate median survival ofabout 3 years (1-3).

Most targeted cancer therapies today delay but rarely prevent tumorprogression. As tumor cells are genomically unstable, they eventuallyacquire mutations and genetic alterations that allow them to evade thetherapy and develop resistance. The rate of killing that is elicited bytargeted agents is too slow, providing the tumors with sufficient timeto adapt to the constant pressure exerted on them by the therapy.Additionally, tumors are heterogeneous and possess a number of differentsubpopulations. Targeted therapies usually target only some of thesesubpopulations and not others, and therefore cannot be expected toeradicate the entire tumor.

Metastatic CRPC typically presents a unique cell surface molecule thatcan be exploited for targeted therapy: prostate-specific membraneantigen (PSMA). PSMA is over-expressed at levels of up to 1000-fold atall Gleason scores (4), while over-expression increases with tumorprogression (5,6). Despite the heterogeneous nature of the disease,primary tumors or metastases that are completely PSMA-negative are rare(7). While the above findings support the notion that PSMA is a highlypromising therapeutic target, no PSMA-targeted therapies are currentlyapproved for clinical use. However, few agents are in clinical trials(8-11).

Thus a continuing need exists for targeted therapy for CRPC.

SUMMARY OF THE INVENTION

The present invention is directed to an improved approach to thetargeting of dsRNA to cancer cells, namely, the generation of a chimericprotein molecule that can deliver dsRNA to e.g. EGFR over-expressingcells. As a non-limiting example, the chimeric protein, dsRBEC(dsRBD-EGF-Chimera) is composed of the dsRNA-binding domain (dsRBD) ofhPKR (residues 1-197) fused via a linker to hEGF (FIG. 7). The dsRBD ofhPKR is composed of two copies of a dsRNA binding motif (dsRBM), whichare connected through a flexible linker and can interact with dsRNA in anon-sequence specific manner [43, 44]. This dsRBD is the polyIC bindingmoiety of dsRBEC. EGF is used as a targeting moiety, which selectivelybinds EGFR and induces endocytosis. As compared with the polymericchemical vector, the chimeric protein is precisely defined and can beproduced simply and inexpensively. Unlike current anti-EGFR therapies,which target the activity of the receptor, our therapy does not inhibitthe EGFR pathway but exploits the over-expression of the receptor forselectivity and for cellular entry.

Additionally described herein is an improved approach to the targetingof dsRNA to cancer cells, namely, the generation of a chimeric proteinmolecule that can deliver dsRNA to PSMA over-expressing cells.

The present disclosure provides a chimeric recombinant protein andencoding nucleic acids thereof which includes a double stranded RNA(dsRNA) binding domain; and a target binding moiety that binds toprostate surface membrane antigen (PSMA).

Additionally described is a complex that includes a described chimericrecombinant protein and dsRNA. Also described are uses of the describedcomplexes in treatment of prostate cancer or inhibition of thedevelopment of tumor neovasculature, and corresponding methods oftreatment of prostate cancer or inhibition of tumor neovasculature thatinclude administering to a subject in need thereof a therapeuticallyeffective amount of the described complex.

The foregoing and other objects, features, and advantages will becomemore apparent from the following detailed description, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E: GFP-SCP binds and selectively internalizes into PSMAover-expressing cells. FIG. 1A: Schematic representation of GFP-SCP.FIG. 1B: LNCaP, PC3 and MCF7 cells were incubated with 25 nM GFP-SCP for5 hr. The cells were fixed and stained with anti-GFP antibody (Cy3) and4, 6-diamidino-2-phenylindole and viewed by laser scanning confocalmicroscopy. FIG. 1C: LNCaP and MCF7 cells were incubated with GFP-SCP,then subjected to flow cytometric analysis. FIG. 1D: LNCaP cells weremonitored by laser confocal imaging, 0 to 72 min after the addition of200 nM GFP-SCP. Sulforhodamine-B was added to the medium immediatelybefore adding the GFP-SCP, to mark the outside of the cells. FIG. 1E:shows GFP fluorescence inside the cell, as measured using ImageJ.

FIGS. 2A-2C: Design, expression and purification of dsRB-SCP. FIG. 2A:Schematic representation of dsRB-SCP. FIG. 2B: Expression andpurification of dsRB-SCP: L: Cleared lysate, M: Molecular weight marker,E1: Eluate following IMAC (nickel sepharose column), E2: PurifieddsRB-SCP eluted from IEX (Ion exchange column). Dashed lines indicatewhere the picture of the gel was cut and reorganized. FIG. 2C: Bindingof dsRB-SCP to dsRNA: dsRB-SCP (0.5-3 μg) was preincubated with 500 bplong dsRNA and electrophoresed on a 2% agarose gel. M: 100 bp DNAmolecular weight marker.

FIGS. 3A-3C: dsRB-SCP/polyIC selectively induces apoptosis of PSMAover-expressing cells. FIG. 3A: Cells were seeded in triplicate, grownovernight, and treated as indicated for 100 hr. Viability was quantifiedusing the CellTiter-Glo Luminescent Cell Viability Assay (Promega). FIG.3B: Surviving cells remained permanently arrested. Cells were seeded intriplicate, grown overnight, and treated as indicated. Medium wasreplaced and viability was quantified after 100/172/344 hr usingCellTiter-Glo. (Control cells were unable to proliferate beyond 2.5doublings because they had reached full confluence). FIG. 3C: LNCaPcells were treated for the indicated times with dsRB-SCP/polyIC orpolyIC alone, lysed and subjected to western blot analysis to detectfull-length and cleaved Caspase-3 and PARP.

FIGS. 4A-4D: dsRB-SCP/polyIC leads to secretion of pro-inflammatorycytokines and recruitment of PBMCs. FIGS. 4A and 4B: LNCaP cells weretreated as indicated for 48 hr, after which medium was collected andIP-10 (FIG. 4B) and RANTES cytokines (FIG. 4A) were measured by ELISAassays. FIG. 4C: LNCaP cells were treated as indicated for 4 h and IFN-βtranscription was measured by qRT-PCR. FIG. 4D: dsRB-SCP/polyIC induceschemotaxis of PBMCs. LNCaP cells were grown and treated as indicated. 48hr after treatment, the cell medium was transferred to the lower chamberof a Transwell chemotaxis plate. PBMCs were added to the upper chamber,and the plates were incubated for 3.5 hr. Then, medium was collectedfrom the lower chamber and lymphocytes that had migrated to the lowerchamber were quantified by FACS.

FIGS. 5A-5B: dsRB-SCP/polyIC induces direct and PBMC-mediated bystandereffects. FIG. 5A: LNCaP-Luc/GFP cells were treated as indicated. After24 hr, PBMC were added to the test wells (black bars), and medium wasadded to control wells (gray bars). Survival of LNCaP-Luc/GFP cells wasmeasured using the Luciferase Assay System (Promega). FIG. 5B:PC3-Luc/GFP+LNCaP: LNCaP cells were treated as indicated. After 24 hr,PC3-Luc/GFPcells were added to the culture. 6 hr later, PBMCs (blackbars) or medium were added to the culture (hatched bars). PC3-Luc/GFP:LNCaP growth medium was treated as indicated. After 24 hr, PC3-Luc/GFPcells were added, and 6 hr later either PBMCs (black bars) or medium(hatched bars) was added. Survival of PC3-Luc/GFP cells was measuredusing the Luciferase Assay System (Promega). T-test indicates highsignificance (***P<0.001).

FIGS. 6A-6C: dsRB-SCP/polyIC treatment together with PBMCs leads to thedestruction of LNCaP spheroids. FIGS. 6A and 6C: Spheroids of R=300-400μm were treated as follows: (a) Untreated, (b) 400 nM dsRB-SCP, (c) 2.5μg/ml polyIC, (d) 400 nM dsRB-SCP+2.5 μg/ml polyIC. Spheroids weretreated four times, on days 1, 2, 4 and 5, and then cultured for 10additional days. Spheroid images were captured by a laser scanningconfocal microscopy at the indicated times; one representative spheroidis shown per treatment. Note the prominent shedding of cells from thetreated spheroid (red arrows). On Day 15, spheroids were labeled withCalcein AM (living cells; green) and Propidium Iodide (dead cells; red).Maximum areas of spheroids, measured using ImageJ, are shown in thegraph (FIG. 6C) (Mean and standard deviation). FIG. 6B: Upper panel:LNCaP-Luc/GFP spheroids treated as indicated. After 24 hr, 8*10⁴ PBMCslabeled with CellTracker™ Violet BMQC (Molecular Probes-LifeTechnologies) were added to the spheroids. Lower panel: PBMC mediumwithout cells was added to the spheroids. Spheroids in both panels werecaptured by laser scanning confocal microscopy 0, 72, 96, 168 hr aftertreatment initiation. Living cells were detected by their GFPfluorescence. PI was added to the spheroids in the lower panel, tohighlight the dead cells. PI staining of upper panel is not shown, asthere is no way to distinguish between dead LNCaP-Luc/GFP cells and deadPBMCs.

FIG. 7: Schematic description of the chimeric protein, dsRBEC. The dsRBDof hPKR enables polyIC binding and is fused via a linker to the homingmoiety, hEGF. The His6 tag facilitates purification on Ni Sepharose.

FIG. 8: 125I-EGF Binding in A431 cells. A431 cells were incubated with0-2,000 pM ¹²⁵I-EGF for 4 hours at 4° C. Total: total binding, NSB:non-specific binding; SB: specific binding. Non-specific binding wasmeasured in the presence of 1 μM unlabeled hEGF. The data were analyzedusing GraphPad Prism 5, yielding a Kd value of 138.3±30.63 pM.

FIG. 9A-9E: Purification of dsRBEC. FIG. 9A) dsRBEC was purified on NiSepharose under native conditions. Samples of total lysate (T), solublefraction (S), unbound fraction (UB) and eluate (EL) were electrophoresedby SDS-PAGE. dsRBEC was visualized with Coomassie dye and by westernblot analysis using an anti-His antibody. FIG. 9B) Samples of BLelectrophoresed on 1% agarose and stained with ethidium bromide.Treatment with 10 μg/ml RNase A eliminated staining. FIG. 9C) dsRBEC waspurified on Ni Sepharose under denaturing conditions (4M urea). Samplesof total lysate (T), soluble fraction (S), unbound fraction (UB) andeluate (EL) were analyzed as in (A). FIG. 9D) Equal amounts of elutedprotein, isolated under native or denaturing conditions, wereelectrophoresed and stained with ethidium bromide. FIG. 9E) SDS-PAGEanalysis of final dsRBEC purification, (15% bis-acrylamide gel stainedwith Coomassie dye) T, total crude lysate before purification; Ni,eluate from 4 ml Ni Sepharose column; S-75, eluate from finalpurification on Superdex75 (uncropped gel can be visualized in FIG. 10).

FIG. 10A-10B: Uncropped gels showing dsRBEC purification. FIG. 10A) NiSepharose column. Lane 1, insoluble pellet following bacterial lysis;Lane 2, soluble lysate before purification (=T in FIG. 9E); Lane 3,unbound protein; Lane 4, molecular weight markers; Lanes 5-13 and 15fractions eluted from Ni Sepharose (Lane 8=Ni in FIG. 9E), Lane 14, Poolof fractions represented in 8-11. FIG. 10B) Superdex75 gel filtration.Pooled fractions 8 through 11 were loaded onto 320 ml Superdex 75. Lane1, same as lane 14 in gel A. Lanes 2-15 eluates collected from thecolumn. The eluates in Lanes 7-11 were pooled, divided into aliquots andused for subsequent analysis. (Lane 8=8-75 in FIG. 9E).

FIG. 11A-11C: Analysis of dsRBEC activity. FIG. 11A) EMSA showingreduced mobility in 2% agarose of polyIC/dsRBEC complexes. Lane 1,polyIC (0.5n); Lanes 2-5, polyIC (0.5n) pre-incubated with dsRBEC (0.5-4μg). FIG. 11B) Displacement of ¹²⁵I-EGF by dsRBEC (▪) or unlabeled hEGF(▴) in A431 cells. The graph shows means±SD from a representativeexperiment, performed using duplicate samples. The Kd was calculated asthe mean from three independent experiments. FIG. 11C) Western blotanalysis of EGFR phosphorylation following treatment of MDA-MB-468 cellswith dsRBEC at various concentrations for 15 minutes.

FIG. 12A-12D: dsRBEC selectively introduces polyIC into EGFRover-expressing cells. FIG. 12A) Expression of EGFR in MDA-MB-468 andMCF7 cells was evaluated by FACS or FIG. 12D) by western blot asdescribed in the Materials and Methods. FIG. 12B) Confocal live imagingof Cy3-polyIC internalization in MDA-MB-468 and MCF7 cells. Cy3-polyICwas delivered by dsRBEC (Upper row), or added directly to the cellculture medium (lower row). The figure shows Cy3-polyIC localization attime 0 (before treatment) and after 120 minutes of treatment (scale bar20 μm). FIG. 12C) Cy3-polyIC/dsRBEC and AlexaFluor647-transferrin wereadded to MDA-MB-468 cells simultaneously. Endosomal localization ofCy3-polyIC/dsRBEC is indicated by its strong co-localization with therecycling endosomal marker transferrin, 60 minutes after the start oftreatment. Cy3-polyIC (red), transferrin (green), merge (yellow). (Scalebar 10 μm).

FIG. 13A-13C: PolyIC/dsRBEC induces apoptosis in MDA-MB-468 but not inMCF7 cells. FIG. 13A) Survival following treatment with dsRBEC alone,polyIC alone or polyIC/dsRBEC for 72 hours was analyzed using methyleneblue. Viable cells are presented as percent of vehicle-treated control(0). Upper x-axis shows concentration of dsRBEC; lower x-axis showsconcentration of polyIC. For MDA-MB-468 cells, the difference betweentreatment with PolyIC/dsRBEC vs PolyIC alone, or vs dsRBEC alone, orversus vehicle was significant (P<0.0001) for all concentrations tested,but is shown in the figure only for 1 μg/ml polyIC, for ease ofpresentation. FIG. 13B) Apoptosis in MDA-MB-468 cells was evaluated bywestern blot showing cleaved PARP and caspase 3 following treatment withdsRBEC alone, polyIC alone or polyIC/dsRBEC for 4 hours. FIG. 13C)MDA-MB-468 and MCF7 cells were treated with dsRBEC alone, polyIC aloneor polyIC/dsRBEC for 8 hours. Annexin V-FITC and PI binding weremeasured by flow cytometry. The percentage of stained cells is writtenin the right corner of the relevant quadrant.

FIG. 14A-14H: PolyIC/dsRBEC induces the expression and secretion ofpro-inflammatory cytokines in MDA-MB-468 cells. FIG. 14A)-D) qRT-PCRanalysis of IFN-beta, CCLS, IP-10 and TNF-alpha, mRNA expressionfollowing treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for2 and 4 hours. Data were normalized to GAPDH and are expressed as foldchange relative to vehicle-treated samples. A representative experimentout of 3 experiments is shown. Error bars represent RQ max. FIG. 14E)-H)Protein levels of IFN-beta, CCLS, IP-10 and TNF-alpha were measured byELISA following treatment with dsRBEC alone, polyIC alone orpolyIC/dsRBEC for hours. Values are averages of triplicate biologicalsamples from one representative experiment. (***, P<0.0001 for effect ofpolyIC/dsRBEC vs polyIC alone, for polyIC/dsRBEC vs dsRBEC alone and forpolyIC/dsRBEC vs vehicle).

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic and/or amino acid sequences provided herewith are shownusing standard letter abbreviations for nucleotide bases, and threeletter code for amino acids, as defined in 37 C.F.R. 1.822. Only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. The Sequence Listing is submitted as an ASCII text file namedSeqList_3152_1_2.txt created Dec. 12, 2016, about 21 KB, which isincorporated by reference herein. In the Sequence Listing:

-   -   SEQ ID NO: 1 is the amino acid sequence of the GFP-SCP protein.    -   SEQ ID NO: 2 is a nucleic acid sequence encoding the GFP-SCP        protein.    -   SEQ ID NO: 3 is the amino acid sequence of the dsRB-SCP protein.    -   SEQ ID NO: 4 is a nucleic acid sequence encoding the dsRB-SCP        protein.    -   SEQ ID NO: 5 is amino acid sequence of the the Arg9 linker        peptide.    -   SEQ ID NOs 6 and 7 are forward and reverse oligonucleotide        primers for IFN-r3 quantification.    -   SEQ ID NOs 8 and 9 are forward and reverse oligonucleotide        primers for GAPDH quantification.    -   SEQ ID NO: 10 is the nucleic acid sequence of the SCP-N primer.    -   SEQ ID NO: 11 is the nucleic acid sequence of the SCP-C primer.    -   SEQ ID NO: 12 is the nucleic acid sequence of the GFP-N primer.    -   SEQ ID NO: 13 is the nucleic acid sequence of the GFP-C primer.    -   SEQ ID NO: 14 is the nucleic acid sequence of the dsRB-N primer.    -   SEQ ID NO: 15 is the nucleic acid sequence of the dsRB-C primer.    -   SEQ ID NO: 16 is the nucleic acid sequence of the 9ARG1 primer.    -   SEQ ID NO: 17 is the nucleic acid sequence of the 9ARG2 primer.    -   SEQ ID NO: 18 is the amino acid sequence of PKR dsRNA.    -   SEQ ID NO: 19 is the nucleic acid sequence of PKR dsRNA.    -   SEQ ID NO: 20 is the amino acid sequence of ScFvJ591.    -   SEQ ID NO: 21 is the nucleic acid sequence of ScFvJ591.    -   SEQ ID NO: 22 is the amino acid sequence of GE11.    -   SEQ ID NO: 23 is the amino acid sequence of the spacer peptide.    -   SEQ ID NO: 24 is the nucleic acid sequence of a 3′ sequence of        the dsRBD.    -   SEQ ID NOs: 25 and 26 are the nucleic acid sequence and amino        acid sequence of the linker.    -   SEQ ID NOs: 27-36 are the nucleic acid sequences of qRT-PCR        primer sequences.

DETAILED DESCRIPTION I. Terms

Unless otherwise noted, technical terms are used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. The singular terms “a,” “an,” and “the”include plural referents unless context clearly indicates otherwise.Similarly, the word “or” is intended to include “and” unless the contextclearly indicates otherwise. It is further to be understood that allbase sizes or amino acid sizes, and all molecular weight or molecularmass values, given for nucleic acids or polypeptides are approximate,and are provided for description. Although methods and materials similaror equivalent to those described herein can be used in the practice ortesting of this disclosure, suitable methods and materials are describedbelow. The term “comprises” means “includes.” The abbreviation, “e.g.”is derived from the Latin exempli gratia, and is used herein to indicatea non-limiting example. Thus, the abbreviation “e.g.” is synonymous withthe term “for example.”

In case of conflict, the present specification, including explanationsof terms, will control. In addition, all the materials, methods, andexamples are illustrative and not intended to be limiting.

Administration: The introduction of a composition into a subject by achosen route. Administration of an active compound or composition can beby any route known to one of skill in the art. Administration can belocal or systemic. Examples of local administration include, but are notlimited to, topical administration, subcutaneous administration,intramuscular administration, intrathecal administration, In addition,local administration includes routes of administration typically usedfor systemic administration, for example by directing intravascularadministration to the arterial supply for a particular organ. Thus, inparticular embodiments, local administration includes intra-arterialadministration and intravenous administration when such administrationis targeted to the vasculature supplying a particular organ. Localadministration also includes the incorporation of active compounds andagents into implantable devices or constructs, such as vascular stentsor other reservoirs, which release the active agents and compounds overextended time intervals for sustained treatment effects.

Systemic administration includes any route of administration designed todistribute an active compound or composition widely throughout the bodyvia the circulatory system. Thus, systemic administration includes, butis not limited to intra-arterial and intravenous administration.Systemic administration also includes, but is not limited to, topicaladministration, subcutaneous administration, intramuscularadministration, or administration by inhalation, when suchadministration is directed at absorption and distribution throughout thebody by the circulatory system.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region, which specificallyrecognizes and binds an epitope of an antigen, such as PSMA. Antibodiesare composed of a heavy and a light chain, each of which has a variableregion, termed the variable heavy (VH) region and the variable light(VL) region. Together, the VH region and the VL region are responsiblefor binding the antigen recognized by the antibody. As used herein,“antibody” includes intact immunoglobulins and the variants and portionsof them well known in the art, such as Fab′ fragments, F(ab)′2fragments, single chain Fv proteins (“scFv”), and disulfide stabilizedFv proteins (“dsFv”). The term also includes recombinant forms such aschimeric antibodies (for example, humanized murine antibodies),heteroconjugate antibodies (such as, bispecific antibodies).

Chimera: A nucleic acid sequence, amino acid sequence, or protein thatcomprises nucleic acid sequence, amino acid sequence, or protein fromtwo or more sources, for example amino acid sequence from two or moredifferent species. In general, chimeric sequences are the result ofgenetic engineering.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked, for example the expression of a nucleic acidencoding the chimeric recombinant proteins described herein. Expressioncontrol sequences are operatively linked to a nucleic acid sequence whenthe expression control sequences control and regulate the transcriptionand, as appropriate, translation of the nucleic acid sequence. Thusexpression control sequences can include appropriate promoters,enhancers, transcription terminators, a start codon (ATG) in front of aprotein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons.

Functional fragments and variants of a polypeptide: Included are thosefragments and variants that maintain one or more functions of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated without materially altering oneor more the polypeptide's functions, including variants of 60%-99%sequence identity to the wildtype or parent polypeptide. First, thegenetic code is well-known to be degenerate, and thus different codonsencode the same amino acids. Second, even where an amino acidsubstitution is introduced, the mutation can be conservative and have nomaterial impact on the essential functions of a protein. Third, part ofa polypeptide chain can be deleted without impairing or eliminating allof its functions. Fourth, insertions or additions can be made in thepolypeptide chain for example, adding epitope tags, without impairing oreliminating its functions. Functional fragments and variants can be ofvarying length. For example, some fragments have at least 10, 25, 50,75, 100, or 200 amino acid residues. Conservative amino acidsubstitution tables providing functionally similar amino acids are wellknown to one of ordinary skill in the art. The following six groups areexamples of amino acids that are considered to be conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Linker: One or more nucleotides or amino acids that serve as a spacerbetween two molecules, such as between two nucleic acid molecules or twopeptides.

Mimetic: A mimetic is a molecule that mimics the activity of anothermolecule, such as a biologically active molecule. Biologically activemolecules can include chemical structures that mimic the biologicalactivities of a compound.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Therapeutically effective amount: A quantity of compound sufficient toachieve a desired effect in a subject being treated. An effective amountof a compound may be administered in a single dose, or in several doses,for example daily, during a course of treatment. However, the effectiveamount will be dependent on the compound applied, the subject beingtreated, the severity and type of the affliction, and the manner ofadministration of the compound.

II. Chimeras of Target-Binding Domain and Double Stranded RNA BindingDomain and their Use in Targeting Double Stranded RNA to Cancer Cells

In one aspect, the present invention provides a recombinant proteincomprising a double stranded RNA (dsRNA) binding domain and atarget-binding moiety. dsRNA-binding domains may readily be identifiedin a peptide sequence using methods available to the average personskilled in the art, such as, but not limited to, the method disclosed inU.S. Pat. No. 6,004,749 (incorporated by reference as if fully disclosedherein).

In certain embodiments, the dsRNA binding domain and the target-bindingmoiety are connected by a spacer peptide. In certain embodiments, therecombinant protein may further comprise a cytolytic peptide.

For purification purposes, the recombinant protein may further comprisea purification tag, such as His6 tag.

The dsRNA binding domain of any one of the recombinant proteins of theinvention may comprise one or more double-stranded RNA-binding motif(dsRBM), i.e. an alpha-beta-beta-beta-alpha fold.

In certain embodiments, said one or more dsRBM is selected from a dsRBMof dsRNA dependent protein kinase (PKR), TRBP, PACT, Staufen, NFAR1,NFAR2, SPNR, RHA or NREBP, as taught by Saunders et al., 2003(incorporated by reference as if fully disclosed herein). In particular,the dsRNA binding domain may comprise two dsRBMs of a PKR, optionallyconnected by a flexible linker.

In certain embodiments, the dsRNA binding domain is selected from thedsRNA binding domain of human PKR and said two dsRBMs are connected by aflexible linker; or the full length human PKR.

In certain embodiments, the dsRNA binding domain comprises amino acidresidues 1-197 of human PKR.

In certain embodiments, the target-binding moiety of any one of therecombinant proteins described above comprises (i) a ligand to a cellsurface receptor; (ii) an antibody, such as a humanized antibody; ahuman antibody; a functional fragment of an antibody; a single-domainantibody, such as a Nanobody; a recombinant antibody; and a single chainvariable fragment (ScFv); (ii) an antibody mimetic, such as an affibodymolecule; an affilin; an affimer; an affitin; an alphabody; ananticalin; an avimer; a DARPin; a fynomer; a Kunitz domain peptide; anda monobody; or (iii) an aptamer.

In certain embodiments, the target-binding moiety of any one of therecombinant proteins described above binds a tumor-associated antigen,such as, but not limited to, epidermal growth factor receptor (EGFR),human epidermal growth factor receptor 2 (HER2), prostate surfacemembrane antigen (PSMA), fibroblast growth factor receptor (FGFR),colony stimulating factor 1 receptor (CSF-1R), platelet-derived growthfactor receptors (PDGFR), insulin-like growth factor 1 receptor (IGF-1R)and MET.

In certain embodiments, the target-binding moiety is an EGFR ligand,such as an EGF or the peptide GE11 of the sequence YHWYGYTPQNVI (SEQ IDNO:22); an anti-EGFR antibody, such as an anti-EGFR scFv or a humanizedor human anti-EGFR antibody; or an anti-EGFR affibody. One example of ananti-EGFR antibody that may be used in accordance with the presentinvention is Erbitux (The biologic effects of C225, a chimericmonoclonal antibody to the EGFR, on human prostate carcinoma. Prewett M,Rockwell P, Rockwell R F, Giorgio N A, Mendelsohn J, Scher H I,Goldstein N I. J Immunother Emphasis Tumor Immunol. 1996November;19(6):419-27). Examples of EGF affibodies that may be used inaccordance with the present invention may be found in Friedman M,Nordberg E, Hoiden-Guthenberg I, Brismar H, Adams G P, Nilsson F Y,Carlsson J, Stahl S Phage display selection of Affibody molecules withspecific binding to the extracellular domain of the epidermal growthfactor receptor. Protein Eng Des Sel. 2007 April; 20(4):189-99(incorporated by reference as if fully disclosed herein).

In certain embodiments, the EGF is human EGF and the EGFR is human EGFR.

In certain embodiments, the target-binding moiety is a humananti-epidermal growth factor receptor 2 (HER2) antibody, such as ananti-HER2 scFv or a humanized or human anti-HER2 antibody; or ananti-HER2 affibody. Example of anti-HER2 affibodies that may be used inaccordance with the present invention may be found in Wikman M, SteffenA C, Gunneriusson E, Tolmachev V, Adams G P, Carlsson J, Stahl S.Selection and characterization of HER2/neu-binding affibody ligands.Protein Eng Des Sel. 2004 May; 17(5):455-62 (incorporated by referenceas if fully disclosed herein).

In certain embodiments, the target-binding moiety is a prostate surfacemembrane antigen (PSMA) ligand, such as DUPA or an analog thereof; ananti-P SMA antibody, such as an anti-PSMA scFv or a humanized or humananti-PSMA antibody (e.g. the full length antibody J591; He Liu, PeggyMoy, Sae Kim, Yan Xia, Ayyappan Rajasekaran, Vincent Navarro, BeatriceKnudsen, and Neil H. Bander. Monoclonal Antibodies to the ExtracellularDomain of Prostate-specific Membrane Antigen Also React with TumorVascular Endothelium); or an anti-PSMA afflbody. Cancer Research Volume.57, Issue. 17, pp. 3629-3634)

In certain embodiments, the PSMA is human PSMA.

In certain embodiments, a spacer peptide may connect between the dsRNAbinding domain and the target-binding moiety of any one of therecombinant proteins described above.

In certain embodiments, the spacer peptide is selected from anoligopeptide comprising a protease recognition sequence; ahomo-oligopeptide of a positively charged amino acid (at physiologicalpH), such as arginine; a peptide of the sequenceACSGSACSGSAGNRVRRSVGSSNG (SEQ ID NO:23) or a homolog thereof a cytolyticpeptide; or a combination thereof.

In certain embodiments, the homo-oligopeptide of arginine is Arg8.

In certain embodiments, a cytolytic peptide may be present within thesequence of any one of the recombinant proteins described above.

In certain embodiments, the cytolytic peptide is selected from Melittinor Candidalysin.

In certain embodiments, the cytolytic peptide is positioned in alocation where it does not negatively affect the binding affinity of thetarget-binding moiety to its target or the binding of the dsRNA bindingdomain with the dsRNA, and may therefore be placed within the spacerpeptide or within the N-terminus of the recombinant protein.

In certain embodiments, the recombinant protein of the present inventioncomprises the dsRNA binding domain of human PKR wherein said two dsRBMsare connected by a flexible linker; and a target-binding moiety selectedfrom an anti-human EGFR antibody, an anti-human HER2 antibody and ananti-PSMA antibody, wherein said dsRNA binding domain and saidtarget-binding moiety are connected by an Arg8 spacer peptide.

In certain embodiments, the recombinant protein of the present inventioncomprises the dsRNA binding domain of human PKR wherein the two dsRBMsare connected by a flexible linker; and an anti-human EGFR antibodyconnected by an Arg8 peptide. In certain embodiments, the recombinantprotein of the present invention comprises the dsRNA binding domain ofhuman PKR wherein the two dsRBMs are connected by a flexible linker; andan anti-human HER2 antibody connected by an Arg8 peptide.

In certain embodiments, the recombinant protein of the present inventioncomprises the dsRNA binding domain of human PKR wherein the two dsRBMsare connected by a flexible linker; and an anti-human PSMA antibodyconnected by an Arg8 peptide.

In certain embodiments, any one of the recombinant proteins describedabove is essentially free of RNA.

In another aspect, the present invention is directed to a complexcomprising any one of the recombinant proteins described above anddsRNA. Preferably, the recombinant protein is essentially free ofcontaminating dsRNA remaining from the manufacturing process of therecombinant protein.

In certain embodiments, the dsRNA of the complex is PKR-activatingdsRNA, such as dsRNA comprising a polyinosinic acid strand and apolycytidylic acid strand (poly IC).

In certain embodiments, the poly IC comprises at least 22ribonucleotides in each strand, for example, 85-300 ribonucleotides ineach strand.

In certain embodiments, the dsRNA of the complex comprises at least onsiRNA sequence directed against e.g. a pro-oncogenic protein, such as,but not limited to, Bcl-xl, Bch 2, Mcl-1, Stat3, Pkb/Akt.

In certain embodiments, the complex comprises the dsRNA binding domainof human PKR wherein said two dsRBMs are connected by a flexible linker;and a target-binding moiety selected from an anti-human EGFR antibody,an anti-human HER2 antibody and an anti-PSMA antibody, wherein saiddsRNA binding domain and said target-binding moiety are connected by anArg8 spacer peptide, and said poly IC or siRNA is non-covalentlyassociated with said dsRNA binding domain.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising any one of the complexes described above and apharmaceutically acceptable carrier.

In an additional aspect, the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding any one of therecombinant proteins described above.

In certain embodiments, the sequence is optimized for expression in abacterial or plant host cell, preferably a plant host cell.

In yet an additional aspect, the present invention provides a vectorcomprising at least one control element, such as a promoter andterminator, operably linked to any one of the nucleic acid moleculesdescribed above, wherein said at least one control element is optimizedfor expression in a bacterial or plant host cell, preferably a planthost cell.

In still another aspect, the present invention is directed to a methodfor manufacturing a recombinant protein comprising a dsRNA bindingdomain and a target-binding moiety, comprising expressing any one of thenucleic acid molecules or vectors described above in a bacterial orplant cell and extracting said recombinant protein from said cells.

In certain embodiments, the method further comprises a step of removingcontaminating host cell RNA from and isolating said recombinant protein,for example by contacting said recombinant protein with urea, e. g. 4Murea, and refolding said recombinant protein.

In certain embodiments, the method comprises expressing the nucleic acidmolecule or vector in a plant cell, such as a tobacco or carrot cell,either in suspension or in a whole plant.

In yet a further aspect, the present invention provides a method fortreatment of cancer characterized by expression of a tumor-associatedantigen, said method comprising systemically administering to a patientin need any one of the complexes or pharmaceutical composition describedabove.

In certain embodiments, the cancer is selected from a cancercharacterized by EGFR-over-expressing cells, a cancer characterized byHER2-overexpressing cells and prostate cancer.

In certain embodiments, the cancer characterized by EGFR-overexpressingcells is selected from non-small-cell-lung-carcinoma, breast cancer,glioblastoma, head and neck squamous cell carcinoma, colorectal cancer,adenocarcinoma, ovary cancer, bladder cancer or prostate cancer, andmetastases thereof.

In certain embodiments, the cancer characterized by HER2-overexpressingcells is selected from breast cancer, ovarian cancer, stomach cancer,and aggressive forms of uterine cancer, such as uterine serousendometrial carcinoma.

In certain embodiments, the cancer characterized by HER2-overexpressingcells is Herceptin/trastuzumab resistant cancer.

In certain embodiments, the complex administered to said patientcomprises the dsRNA binding domain of human PKR wherein said two dsRBMsare connected by a flexible linker; and a target-binding moiety selectedfrom an anti-human EGFR antibody, an anti-human HER2 antibody and ananti-PSMA antibody, wherein said dsRNA binding domain and saidtarget-binding moiety are connected by an Arg8 spacer peptide, and saidpoly IC or siRNA is non-covalently associated with said dsRNA bindingdomain.

In certain embodiments, the method further comprises administeringimmune cells, such as tumor-infiltrating T-cells (T-TILs), tumorspecific engineered T-cells, or peripheral blood mononuclear cells(PBMCs).

III. Overview of Several Embodiments

Described herein is a chimeric recombinant protein which includes adouble stranded RNA (dsRNA) binding domain; and a target binding moietythat binds to prostate surface membrane antigen (PSMA).

In particular embodiments, the chimeric recombinant protein furtherincludes a spacer peptide between the dsRNA binding domain and thetarget binding moiety.

In some embodiments, dsRNA binding domain of the chimeric recombinantprotein includes at least one double-stranded RNA-binding motif (dsRBM),such as a dsRBM of dsRNA dependent protein kinase (PKR), TRBP, PACT,Staufen, NFAR1, NFAR2, SPNR, RHA, or NREBP. In one example the at leastone dsRBM includes a polypeptide sequence at least 70% identical toamino acids 1-197 of human PKR as set forth as SEQ ID NO: 18.

In particular embodiments of the chimeric recombinant protein, thetarget binding moiety is a polypeptide, antibody, antibody fragment, orantibody mimetic.

In other particular embodiments of the chimeric recombinant protein, thespacer peptide is selected from an oligopeptide comprising a proteaserecognition sequence; a homo-oligopeptide of a positively charged aminoacids; and a combination thereof. In one examplea, the spacer peptide isa homo-oligopeptide of arginine.

In a particular embodiment of the described chimeric recombinantprotein, the double stranded RNA (dsRNA) binding domain is at least onedsRNA binding domain of human PKR as set forth in SEQ ID NO: 18, or afunctional variant thereof, the spacer peptide is ARG9 as set forth inSEQ ID NO: 5, or a functional variant thereof, and the target bindingmoiety is a single chain anti-PSMA antibody as set forth in SEQ ID NO:20, or a functional variant thereof.

In another particular embodiment, the chimeric recombinant proteinincludes a polypeptide at least 70% identical to the sequence set forthas SEQ ID NO: 3.

Additionally described herein is a complex which includes the describedchimeric recombinant protein and dsRNA, such as a dsDNA including apolyinosinic acid strand and a polycytidylic acid strand (poly IC).

In particular embodiments, the described complexes are used in treatmentof prostate cancer or inhibition of the development of tumorneovasculature, such as in methods of treatment for prostate cancer orinhibition of tumor neovasculature which include administering to asubject in need thereof a therapeutically effective amount of thedescribed complex thereby treating the cancer or inhibiting growth oftumor neovasculature.

In some embodiments of the described methods, the complex isadministered systemically or locally. In other embodiments, the methodsfurther include administering to the subject a therapeutically effectiveamount of peripheral blood mononuclear cells (PBMCs).

Further described herein are nucleic acids that encode any of thedescribed chimeric recombinant proteins.

In particular embodiments, the described nucleic acid sequences areoptimized for expression in a bacterial or plant host cell.

III. Chimeric Polypeptides for Targeting dsRNA to PSMA-Expressing Cells

Described herein are chimeric recombinant polypeptides that can be usedto target dsRNA to a cell expressing prostate-specific membrane antigen(PSMA). The described chimeric recombinant polypeptides include at leasta dsRNA binding domain and a domain (also referred to herein as amoiety) that specifically targets PSMA. In particular embodiments, thedescribed polypeptides also include a linker between the dsRNA bindingdomain and the target binding domain. Functional variants of thechimeric recombinant polypeptides are also described.

dsRNA-binding domains may readily be identified in a peptide sequenceusing methods available to the average person skilled in the art. ThedsRNA binding domain of any one of the described recombinant proteinsmay include one or more double-stranded RNA-binding motif (dsRBM), suchas an alpha-beta-beta-beta-alpha fold.

In certain embodiments said one or more dsRBM is selected from a dsRBMof dsRNA dependent protein kinase (PKR), TRBP, PACT, Staufen, NFAR1,NFAR2, SPNR, RHA or NREBP. In particular, the dsRNA binding domain maycomprise two dsRBMs of a PKR, optionally connected by a flexible linker.

In a particular embodiment, the dsRNA binding domain is the dsRNAbinding domain of human dsRNA dependent protein kinase (PKR), or afunctional variant thereof, including a polypeptide that shares about60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity with theamino acid sequence set forth herein as SEQ ID NO: 18.

In certain embodiments, the target-binding moiety of any one of therecombinant proteins described herein includes (i) a ligand to a cellsurface receptor; (ii) an antibody, such as a humanized antibody; ahuman antibody; a functional fragment of an antibody; a single-domainantibody, such as a Nanobody; a recombinant antibody; and a single chainvariable fragment (ScFv); or (iii) an antibody mimetic, such as anaffibody molecule; an affilin; an affimer; an affitin; an alphabody; ananticalin; an avimer; a DARPin; a fynomer; a Kunitz domain peptide; anda monobody,

In certain embodiments, the target-binding moiety is a prostate surfacemembrane antigen (PSMA) ligand, such as DUPA or an analog thereof; ananti-PSMA antibody, such as an anti-PSMA scFv or a humanized or humananti-PSMA antibody (e.g. the full length antibody J591); or an anti-PSMAaffibody.

In a particular embodiment, the PSMA targeting moiety is a single chainantibody against PSMA, ScFvJ591, or a functional variant thereof,including a polypeptide that shares about 60%, 70%, 75%, 80%, 85%, 90%,95%, or 98% sequence identity with the amino acid sequence set forthherein as SEQ ID NO: 19.

The described spacer peptide can be any oligopeptide known in the artfor connecting two functional domains of a polypeptide chimera. Incertain embodiments, the spacer peptide (linker) includes anoligopeptide comprising a protease recognition sequence; or ahomo-oligopeptide of a positively charged amino acid (at physiologicalpH), such as arginine.

In a particular embodiment, the linker (spacer peptide) between thedsRNA binding domain and the target binding moiety is the ARG9 peptide,or a functional variant thereof, including a polypeptide that sharesabout 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity withthe amino acid sequence set forth herein as SEQ ID NO: 5.

In a particular embodiment, the chimeric recombinant polypeptide is thepolypeptide having the amino acid sequence set forth herein as SEQ IDNO: 3, or a functional variant thereof, including a peptide that sharesabout 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity withSEQ ID NO: 4.

In other embodiments the variation from the described sequence can beconservative substitutions that one of skill will not expect tosignificantly alter the shape or charge of the polypeptide. Thedescribed polypeptides also include those polypeptides that share 100%sequence identity to those indicated, but which differ inpost-translational modifications from the native or natively-producedsequence.

In particular embodiments, the described recombinant polypeptides areprovided as a discrete biomolecules. In other embodiments, the describedpolypeptides are a domain of a larger polypeptide, such as anindependently-folded structural domain, or an environment-accessiblefunctional domain.

Additionally described herein is a complex that includes any one of thedescribed recombinant proteins and dsRNA. In certain embodiments, thedsRNA of the complex is PKR-activating dsRNA, such as dsRNA comprising apolyinosinic acid strand and a polycytidylic acid strand (poly IC). Incertain embodiments, the poly IC includes at least 22 ribonucleotides ineach strand, for example, 85-300 ribonucleotides in each strand. Incertain embodiments, the dsRNA of the complex comprises at least onesiRNA sequence directed against a pro-oncogenic protein, such as, butnot limited to, Bcl-xl, Bcl-2, Mcl-1, Stat3, Pkb/Akt.

In particular embodiments, the described complexes are a component of apharmaceutical composition that includes a pharmaceutically acceptablecarrier as described above.

Also provided herein are nucleic acids encoding the described chimericrecombinant polypeptides, such as the nucleic acid set forth herein asSEQ ID NO: 4.

It will be appreciated that due to degeneracy of the genetic code, thesequences of the described nucleic acids can vary significantly from thesequence set forth herein as SEQ ID NO 4; and without any change in theencoded polypeptide. Other and/or additional mutations in the describedpolypeptides, such as conservative amino acid mutations, can also beincluded without an appreciable difference. Accordingly, in someembodiments, the described nucleic acids share between 60%-100% sequenceidentity with SEQ ID NO 4, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, or98% sequence identity. In a particular example, the nucleic acidsequence is adjusted to account for natural codon bias in a particularorganism such as a bacterial or plant cell. Such adjustments are knownto the art, and can be found (12)

In particular embodiments, the described nucleic acid sequences arecontained within a DNA cloning and/or expression plasmid as are standardin the art. It will be appreciated that any standard expression plasmidcan be used to express one or more of the described chimericpolypeptide-encoding nucleic acids. Such plasmids will minimally containan origin of replication, selection sequence (such as, but not limitedto an antibiotic resistance gene), and expression control sequencesoperably linked to the described nucleic acid. In particularembodiments, the expression plasmids include post-translationalsequences (e.g. signal sequences to direct polypeptide processing andexport) that are encoded in-frame with the described nucleic acids. Inparticular embodiments, the expression control sequences are those knownto the art for optimized expression control in a bacterial or planthost.

Particular non-limiting examples of bacterial expression plasmidsinclude IPTG-inducible plasmids, arabinose-inducible plasmids and thelike. Other non-limiting examples of expression induction include lightinduction, temperature induction, nutrient-induction, and autoinduction,plant and mammalian-specific DNA expression plasmids. Custom-madeexpression plasmids are commercially available from suppliers such asNew England Biolabs (Ipswich, Mass.) and DNA 2.0 (Menlo Park, Calif.).

In particular embodiments, the described polypeptides can be formulatedfor immediate release, whereby they are immediately accessible to thesurrounding environment, thereby providing an effective amount of theactive agent(s), upon administration to a subject, and until theadministered dose is metabolized by the subject.

In yet another embodiment, the described polypeptides can be formulatedin a sustained release formulation or system. In such formulations, thetherapeutic agents are provided for an extended duration of time, suchas 1, 2, 3, 4 or more days, including 1-72 hours, 24-48 hours, 16-36hours, 12-24 hours, and any length of time in between. In particularembodiments, sustained release formulations are immediately availableupon administration, and provide an effective dosage of the therapeuticcomposition, and remain available at an effective dosage over anextended period of time. In other embodiments, the sustained releaseformulation is not immediately available within the subject and onlybecomes available, providing a therapeutically effective amount of theactive compound(s), after the formulation is metabolized or degraded soas to release the active compound(s) into the surrounding environment.

In one embodiment, a pump may be used. In another embodiment, thesustained released formulations include polymeric materials commonlyused in the art, such as in implants, gels, capsules, and the like.

Therapeutic preparations will contain a therapeutically effective amountof at least one active ingredient, preferably in purified form, togetherwith a suitable amount of carrier so as to provide proper administrationto the patient. The formulation should suit the mode of administration.

IV. Methods of Treatment of PSMA-Associated Diseases

PSMA expression is associated with cancerous cells, particularlyprostate cancer and tumor-associated neovasculature (13). In yet afurther aspect, the present disclosure provides a method for treatmentof cancer characterized by expression of a PSMA, said method byadministering to a subject in need thereof, any one of the complexes orpharmaceutical composition described herein.

In some embodiments, the described complex is administered to thesubject in combination with other pharmaceutical agents for treatment ofthe cancer under treatment. For example, in particular examples ofcancer treatment, administration of the described can be combined withsurgery, cell therapy, chemotherapy and/or radiation therapy. The one ormore therapies in combination with the described polypeptides can beadministered to the subject in sequence (prior to or following) orconcurrently with the described polypeptides. Where applicable, inparticular embodiments, combinations of active ingredients can beadministered to a subject in a single or multiple formulations, and bysingle or multiple routes of administration. In particular embodiments,the methods of treatment include the sequential or concurrentadministration of peripheral blood mononuclear cells (PBMCs).

The amount of each therapeutic agent for use in the described methods,and that will be effective, will depend on the nature of the cancer tobe treated, as well its stage of the disorder or condition.Therapeutically effective amounts can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and should be decided accordingto the judgment of the health care practitioner and each patient'scircumstances. The specific dose level and frequency of dosage for anyparticular subject may be varied and will depend upon a variety offactors, including the activity of the specific compound, the metabolicstability and length of action of that compound, the age, body weight,general health, sex, diet, mode and time of administration, rate ofexcretion, drug combination, and severity of the condition of the hostundergoing therapy.

The therapeutic compounds and compositions of the present disclosure canbe administered at about the same dose throughout a treatment period, inan escalating dose regimen, or in a loading-dose regime (e.g., in whichthe loading dose is about two to five times the maintenance dose). Insome embodiments, the dose is varied during the course of a treatmentbased on the condition of the subject being treated, the severity of thedisease or condition, the apparent response to the therapy, and/or otherfactors as judged by one of ordinary skill in the art. In someembodiments long-term treatment with the drug is contemplated.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1: Methods

Cloning of GFP-SCP and dsRB-SCP

Plasmids pGFP-SCP (encoding GFP linked via Arg9 to the single chainantibody, ScFvJ591, against PSMA; 56 kDa) and psRB-SCP (encoding dsRB ofhuman PKR linked via Arg9 to ScFvJ591; 48 kDa) (FIG. 1A) wereconstructed as follows:

SCP (single chain antibody against PSMA, ScFvJ591) was amplified by PCRfrom plasmid SFG-Pz1 (14), using primers SCP-N and SCP-C. GFP wasamplified by PCR from plasmid pEGFP-N3 (Clontech), using primers GFP-Nand GFP-C. dsRB was amplified by PCR from plasmid DRBM-DT-EGF (15) usingprimers dsRB-N and dsRB-C. To prepare the Arg9 linker (GSRRRRRRRRGRKA;SEQ ID NO: 5), oligonucleotide 9ARG1 was annealed to its complementaryoligonucleotide 9ARG2. The oligonucleotides used are listed in Table 1.GFP-SCP was constructed in stages in the bacterial expression vectorpET28a (Novagen): GFP was cloned after the His₆ tag of plasmid pET28a,between the Ndel and BamHI restriction sites, SCP was cloned between theHindIII and XhoI sites, and the Arg9 linker was inserted between theBamH1 and HindIII sites, to give the fusion His₆-GFP-Arg9-SCP (FIG. 1A).For the construction of dsRB-SCP, the GFP fragment was replaced with thedsRB sequence using restriction sites Ndel and BamHI to give the fusionHis₆-dsRB-Arg9-SCP (FIG. 2A). The expected sequences were confirmed atThe Center for Genomic Technologies at The Hebrew University ofJerusalem (Supplementary).

TABLE 1 Oligonucleotides used for the construction of pGFP-SCP andpsRB-SCP. Name Sequence 5′ to 3′ SCP-N TTTACTCGAGCGGAGGTGCAGCTGCAGC(SEQ ID NO: 10) SCP-C TTTTGCTCAGCGCCGTTACAGGTCCAGC CATG (SEQ ID NO: 11)GFP-N TTTTCATATGGTGAGCAAGGGCG (SEQ ID NO: 12) GFP-CTAAGGATCCGCCACCGCCGCTTTT CTTGTACAGC(SEQ ID NO: 13) dsRB-NTTTCATATGATGGCTGGTGATC (SEQ ID NO: 14) dsRB-CTTAGGATCCGCCACCGCCGCTCTCCGA TAAGATCTGCAG (SEQ ID NO: 15) 9ARG1GATCCCGTCGTCGCCGTCGTCGCCGTC GCGGCCGCAA (SEQ ID NO: 16) 9ARG2AGCTTTGCGGCCGCGACGGCGACGACG GCGACGACGG (SEQ ID NO: 17)Expression of GFP-SCP and dsRB-SCP

The chimeric proteins were expressed in E. coli BL21trxB(DE3) (Novagen)which had been transformed with plasmid pRARE, which encodes tRNAs forrare codons. The bacteria were grown at 37° C., in 2xYT medium,supplemented with 25 μg/ml chloramphenicol, 30 μg/ml kanamycin, 100μg/ml ampicillin, 1% glucose and 5% NPS buffer (1M KH₂PO₄, 1M Na₂HPO₄,0.5M (NH₄)₂SO₄). When the culture reached OD₆₀₀˜0.3, 0.1% glycerol and0.1 mM L-glutamic acid were added, and the culture was moved to 42° C.,to induce the expression of E. coli chaperones and enhance proteinsolubility. When the culture reached O.D₆₀₀˜0.9, it was cooled down onice and transferred to 14° C. After a 10 min adjustment period, 0.5mmol/L IPTG was added, followed by incubation for 24 h. The bacteriawere harvested and the pellet stored at −80° C. until purification.

Purification of GFP-SCP and dsRB-SCP

GFP-SCP: The pellet obtained from 1.2 L of E. coli BL21trxB(DE3, pRARE,pGFP-SCP) was thawed on ice in 60 ml binding buffer (Buffer A, 30 mMHEPES pH 8.3, 0.5M NaCl, 10% glycerol, 10 mM imidazole) supplementedwith a protease inhibitor cocktail, 3 mg/ml lysozyme and DNase, andlysed using a LV1 microfluidizer (Microfluidics). The extract wasclarified by centrifugation for 30 min (15,000×g, 4° C.), loaded onto an8 ml nickel sepharose FF IMAC column (GE Healthcare), and washed with 10column volumes (CV) of binding buffer, followed by 6 CV of 5% Buffer B(30 mM HEPES pH 8.3, 0.5M NaCl, 10% glycerol, 1M imidazole), 6 CV of 10%Buffer B and 1 CV of 15% Buffer B. The protein was eluted with 60%Buffer B. Fractions containing the chimera (8 ml total) were loaded on a500 ml sephacryl S-200 gel filtration column (GE Healthcare)pre-equilibrated with GF buffer (30 mM HEPES pH 8.3, 0.5M NaCl, 10%glycerol). The fractions eluted after 0.5 CV were pooled, concentratedusing Vivaspin-20 (MWCO: 30000, GE Healthcare) and loaded onto 350 mlsuperdex-75. The fractions eluted after 0.5 CV were subjected toSDS-PAGE and stained with InstantBlue (Expedeon). The fractions thatcontained highly purified chimera were pooled, concentrated usingVivaspin-20 (GE Healthcare), and stored in aliquots at −80° C.

dsRB-SCP: The pellet obtained from 6 L of E. coli BL21trxB(DE3, pRARE,pdsRB-SCP) was thawed in 300 ml binding buffer A supplemented withprotease inhibitors, lysozyme and DNase, lysed and clarified as above.To release bound host nucleic acids, the cleared lysate was mixed 1:1(vol:vol) with 8M urea. The mixture was incubated at 4° C. for 1.5 hrand then loaded onto 60 ml nickel sepharose FF column pre-equilibratedwith buffer C (Buffer A supplemented with 0.5% Tween 80 and 4M urea),and washed with 12.4 CV Buffer C. To refold the protein, a slow lineargradient of Buffer C to Buffer D (Buffer A supplemented with 0.5% Tween80), 10 CV, 0.8 ml/min flow was applied. The column was washed with 3 CVof 10% and 3 CV of 25% Buffer E (30 mM HEPES pH 8.3, 0.5M NaCl, 10%glycerol, 500 mM imidazole, 0.5% Tween 80), and the protein was elutedwith 100% buffer E. The fractions containing the chimera were pooled anddiluted 1:1 with dilution buffer (30 mM MES pH, 10% Glycerol, 0.5%Tween). The diluted protein was clarified by centrifugation for 30 min(15,000×g, 4° C.) and loaded onto a 66 ml Fracto-gel EMD SO3 IEX column(Merck). A manual step gradient (7 CV) of Buffer F (30 mM MES pH, 100 mMNaCl, 10% Glycerol, 0.001% Tween) and 25%, 27%, 30%, 37% and 38% BufferG (30 mM HEPES pH 8.3, 2M NaCl, 10% glycerol, 0.001% Tween 80) wasapplied. Samples of the eluted fractions were subjected to SDS-PAGE andstained with InstantBlue (Expedeon). Fractions that contained purifiedchimera were pooled, concentrated, and stored at −80° C. as above.

Cell Lines

LNCaP cells were cultured in RPMI 1640 medium supplemented with 10 mMHEPES pH 7.4 and 1 mM sodium pyruvate. VCaP cells were cultured in DMEM(Dulbecco's Modified Eagle Medium). PC3 and DU145 cells were cultured inMEM (Minimum Essential Medium) supplemented with 1% non-essential aminoacids, 1 mM sodium pyruvate, 10 mM Hepes pH 7.4 and 1% MEM vitaminmixture. MCF7 cells were cultured in RPMI 1640 medium. All tissueculture media were supplemented with penicillin (100 U/ml), streptomycin(100 mg/1) and 10% FBS (fetal bovine serum). All cell lines werepurchased from the American Type Culture Collection (ATCC), tested andshown to be mycoplasma-free.

LNCaP-Luc/GFP and PC3-Luc/GFP were generated using lentiviral vectorsencoding the fusion gene luciferase-GFP (Luc/GFP) as previouslydescribed (16). PBMCs were isolated from fresh human peripheral blood bystandard Ficoll density-gradient centrifugation (17). All cells wereincubated at 37° C. with 5% CO₂ in a humidified incubator. All cellculture reagents were purchased from Biological Industries, Bet Ha'emek,and Israel.

Flow Cytometry

Cells were plated onto 12-well plates at a density of 1×10⁵ cells perwell, grown for 40 hr and incubated with GFP-SCP. After incubation cellswere trypsinized, washed in PBS, resuspended in lml cold PBS andsubjected to flow cytometry analysis using BD FACS ARIAIII (BDBiosciences, USA) equipped with 488 nm laser. 10,000 cells were acquiredfor each treatment. The cells were gated to include only live cells andthe subpopulation was analyzed for GFP levels. All data was analyzedusing FlowJo software (Becton Dickinson).

Immunocytochemistry

LNCaP, PC3 and MCF7 cells were grown for 48 hr and incubated with 25 nMGFP-SCP for 5 hr at 37° C. After incubation cells were fixed with 4%Paraformaldehyde, washed twice with PBS, permeabilized and stained withgoat anti-GFP antibody (1:1000, Abcam ab5450), followed by incubationwith DyLight 488-conjugated anti-goat secondary antibody (1:300, JacksonImmunoResearch Laboratories). 4, 6-diamidino-2-phenylindole (DAPI) wasused to stain DNA. Stained samples were observed with a confocalmicroscope (FLUOVIEW FV-1000, Olympus, Japan).

Live Cell Imaging

GFP-SCP localization was observed in live LNCaP cells, using time-lapseconfocal microscopy (FLUOVIEW FV-1000, Olympus, Japan). LNCaP cells weregrown for 48 hr in 8-well μ-slides (Ibidi, cat no 80826). After changingthe medium, 200 nM GFP-SCP was added directly to the chamber, the cellswere immediately observed and subsequent images were taken every 6minutes, for 72 mins. The images were analyzed using FLUOVIEW Viewersoftware (Ver.4.2).

dsRNA Electrophoretic Mobility Shift Assay (EMSA)

500 bp long dsRNA transcribed from the control template of theMEGAscript® RNAi Kit (AM1626) was labeled using the Label I T® NucleicAcid Labeling Reagents kit (Mirus). 1 μg of labeled dsRNA was incubatedfor 30 minutes with increasing amounts of purified dsRB-SCP (0.5-3 μg),and the mixture was electrophoresed on a 2% agarose gel. The gel wasvisualized by staining with ethidium bromide.

Preparation of dsRB-SCP/polyIC Complex

PolyIC used for all experiments was low molecular weight (LMW) polyIC(InvivoGen). For all experiments, dsRB-SCP/polyIC, polyIC alone ordsRB-SCP alone was prepared in binding buffer (30 mM HEPES pH 8.3, 0.5MNaCl, 10% glycerol) at the concentrations indicated in the text, andpre-incubated for 45 minutes at room temperature, before addition to thecells.

Survival Assay

LNCaP, VCaP, PC3 and MCF7 cells were seeded in 96-well plates intriplicate (5000 cells/well) and grown overnight. dsRB-SCP/polyIC,polyIC alone or dsRB-SCP was added to the cells, which were thenincubated for additional 100 hr. Survival was measured using theCellTiter-Glo Luminescent Cell Viability Assay (Promega).

For the rescue experiment, LNCaP cells were seeded (5000 cells/well) inthree 96-well plates pre-coated with poly-lysine. For each plate,treatments were repeated in triplicate wells and the cells were grownovernight. The cells were then treated with dsRB-SCP/polyIC, polyICalone or dsRB-SCP alone. The first plate was assayed for survival after100 hr. The medium in the second plate was changed after 100 hr andsurvival was assayed after 172 hr. The medium in the third plate waschanged after 100 hr and again after 172 hr and survival was assayedafter 344 hr.

Immunoblots

LNCaP cells were seeded in 6-well plates (1×10⁶ cells/well), grownovernight and treated with dsRB-SCP/polyIC or polyIC alone at theindicated concentrations. After 7, 16 or 24 hr cells were lysed withboiling Laemmli sample buffer (10% glycerol, 50 mmol/L Tris-HCl, pH 6.8,3% SDS, and 5% 2-mercaptoethanol) and the lysates were then subjected towestern blot analysis (18). The cleavage of PARP and caspase-3 wasmonitored using anti-PARP (cat #95425), anti-caspase3 (cat #96625) andanti-cleaved caspase-3 (cat #96615) (all from Cell SignalingTechnology). As an internal control to normalize the amount of proteinapplied in each lane the blots were also incubated with anti-GAPDH(Santa Cruz, sc-25778).

Detection of Secreted Chemokines (IP-10 and RANTES) by ELISA

LNCaP cells were seeded in 96-well plates in triplicate and grownovernight (10,000 cells/well). Cells were then treated withdsRB-SCP/polyIC or polyIC alone at the indicated concentrations. After48 hr the medium was collected and the concentrations of IP-10 andRANTES were measured using commercial ELISA kits (PeproTech).

RNA Isolation, cDNA Synthesis and Quantitative Real-Time PCR

LNCaP cells were seeded in 24-well plates (500,000 cells per well) andgrown overnight. Cells were then treated for 4 hr with dsRB-SCP/polyIC,polyIC alone or dsRB-SCP alone at the indicated concentrations. Thecells were lysed and total RNA was extracted using the EZ-10 DNA AwayRNA-Miniprep Kit (Bio Basic). Complementary DNA (cDNA) was synthesizedusing the High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems). IFN-β gene expression levels were compared usingquantitative real-time PCR and normalized to GAPDH expression using theΔΔ CT method. The primers used for IFN-β quantification were: forward:5′ ATGACCAACAAGTGTCTCCTCC 3′ (SEQ ID NO: 6) and reverse: 5′GCTCATGGAAAGAGCTGTAGTG 3′ (SEQ ID NO: 7). The primers for GAPDHquantification were forward: 5′ GAGCCACATCGCTCAGAC 3′ (SEQ ID NO: 8) andreverse: 5′ CTTCTCATGGTTCACACCC 3′ (SEQ ID NO: 9).

Chemotaxis of PBMC

LNCaP cells were seeded in 24-well plates pre-coated with poly-lysine(250,000 cells/well) and grown overnight. Then, the medium was replacedby low-serum medium (0.15% FBS) and the cells were treated withdsRB-SCP/polyIC at the indicated concentrations. After 48 hr conditionedmedium was collected from the cells and placed in the bottom well of a24-well Transwell system (microporous polycarbonate membrane (0.5 μm)Corning; Costar). Freshly isolated PBMCs (1×10⁶) in low-serum medium(0.15% FBS) were added to the upper chamber. After 3.5 hr, medium fromthe lower chamber was collected and the migrated cells were quantifiedby FACS analysis, scatter-gating on lymphocytes.

Analysis of Bystander Effects in Co-Culture Systems

In order to measure the viability of a single cell line in co-culturewith other cells, we generated cells that expressed luciferase (eitherLNCaP-Luc/GFP or PC3-Luc/GFP).

The immune-cell-mediated bystander effect was analyzed usingLNCaP-Luc/GFP cells co-cultured with PBMCs: LNCaP-Luc/GFP cells wereseeded in triplicate in 96-well plates pre-coated with poly-lysine(10,000 cells/well) and grown overnight. The cells were then treatedwith dsRB-SCP/polyIC, polyIC alone or dsRB-SCP alone at the indicatedconcentrations. After 24 hr, freshly isolated PBMCs were added to theculture (1×10⁵ per well). 48 hr later, the survival of LNCaP-Luc/GFPcells was measured based on luciferase activity using the LuciferaseAssay System (Promega).

The combined direct and immune-cell-mediated bystander effect wasanalyzed using LNCaP cells co-cultured with PC3-Luc/GFP and PBMCs: LNCaPcells were seeded in triplicate in 96-well plates pre-coated withpoly-lysine (6,000 cells/well) and grown overnight, and the cells weretreated with dsRB-SCP/polyIC, polyIC alone or dsRB-SCP alone. After 16hr PC3-Luc/GFP cells (4,000 cells/well) were added to the culture. 24 hrafter treatment freshly isolated PBMCs (1×10⁵/well) were added to theculture. 48 hr later survival of the PC3-Luc/GFP cells was measuredbased on luciferase activity, using the Luciferase Assay system(Promega).

Tumor Spheroid Model

Tumor spheroids were generated using agar-coated plates. 96-well plateswere coated with 50 μl/well agar (1.5% (wt/vol) dissolved in RPMI)according to ref (19). LNCaP or LNCaP-Luc/GFP cells were seeded (2000cells per well) and incubated. After 97 hr, a single spherical spheroidof R=300-400 μm had formed in each well.

To measure LNCaP spheroids following treatment with dsRB-SCP/polyIC, wetransferred the spheroids individually to 96-well plate (1spheroid/well) pre-coated with a very thin, even layer of polyHEMA (120mg/ml dissolved in 95% ethanol). To transfer the spheroids, we firstlifted each spheroid together with its 200 μl of medium into a 96U-wellplate (with U-shaped wells). The plate was centrifuged for 10 minutes at220 g and the medium was replaced with 80 μl of fresh medium. Thespheroid was then transferred, together with its 80 μl of medium, to thepolyHEMA-coated plate. dsRB-SCP/polyIC, polyIC alone or dsRB-SCP alonewere added at the indicated concentrations. Treatment continued for 5days. On days 1, 2, 4 and 5, half of the medium in each well was removedand replaced with fresh medium containing the appropriate treatment. OnDay 15, spheroids were stained with calcein AM (1:1000, Molecular Probesc3099) and 0.5 μg/ml propidium iodide. Spheroids were monitored usingconfocal microscopy and size was measured using ImageJ software.

To analyze the immune-cell-mediated bystander effects on tumorspheroids, we treated LNCaP-Luc/GFP spheroids once, directly on the agarplate, with dsRB-SCP/polyIC, polyIC alone or dsRB-SCP alone at theindicated concentrations. After 24 hr fresh PBMCs were labeled using 1μM CellTracker™ Violet BMC (Molecular Probes-Life Technologies)according to the manufacturer's protocol. 8×10⁴ PBMCs were added to thespheroid culture. The co-culture was monitored using confocalmicroscopy.

Example 2: Construction and Assay of PSMA-Targeting Chimeric Proteins

ScFvJ591 Selectively Targets PSMA Over-Expressing Prostate Cancer Cellsand Efficiently Delivers its Cargo into the Cells

We first tested whether the single chain antibody ScFvJ591 could be usedas a homing ligand, as part of a chimeric protein. We generatedpGFP-Arg9-ScFvJ591, encoding GFP as a tracking marker fused to thesingle chain antibody against PSMA, ScFvJ591, via a linker comprising anendosomal escape sequence (FIG. 1A). The 56 kDa recombinant protein,GFP-SCP (GFP-Arg9-ScFvJ591), was expressed in E. coli and purified in a3-step purification process comprising affinity purification followed bytwo steps of gel filtration (see Methods).

We tested the selectivity of GFP-SCP using confocal microscopy. Weincubated the chimeric protein with LNCaP cells, which over expressPSMA, and analyzed binding after 5 hr. PC3 and MCF7 cells, which do notexpress PSMA, served as negative controls. The confocal imagesdemonstrated that GFP-SCP bound to LNCaP cells and was selectivelyinternalized, while no binding was evident in PC3 or MCF7 cells (FIG.1B). We next compared uptake of GFP-SCP to LNCaP and MCF7 cells usingflow cytometry. We used two doses of GFP-SCP (200 nM, 400 nM) over twotime periods (30 min, 60 min). The accumulation of GFP-SCP was measuredby the resulting fluorescence shift. As expected, the observedfluorescence levels were correlated with the concentration of GFP-SCPand incubation period (FIG. 1C). These results suggest time-dependentand dose-dependent internalization of GFP-SCP. In contrast, in MCF7cells, which lack PSMA, no accumulation of GFP-SCP was observed (FIG.1C). To monitor the localization of GFP-SCP, we incubated LNCaP cellswith GFP-SCP and observed them using live-cell confocal microscopy.Initially, GFP-SCP fluorescence was confined to the cell surface and nofree diffusion was observed (FIG. 1D, E). Minutes later, GFP-SCP enteredthe cell via endocytosis, as indicated by the appearance of smallintracellular punctate structures (FIG. 1D, E). Over time, thesestructures increased in number. In addition, increased intracellulardiffused powdery fluorescence was observed (FIG. 1D, E), indicating thatthe GFP had escaped from the endosome and diffused to the cytosol. Theaccumulation of the GFP inside the cell increased linearly over thefirst 40 min after binding (FIG. 1D, E).

Design, Expression and Purification of a Chimeric Protein that can Carryand Internalize polyIC Selectively into PSMA Over-Expressing ProstateCancer Cells

Based on the structure of the GFP-SCP chimera, we designed a chimericprotein that would specifically deliver polyIC into PSMA over-expressingcells. We replaced the GFP moiety with the dsRNA-binding domains of PKR(dsRBDs) (FIG. 2A). The chimeric 48 kDa protein, dsRB-SCP(dsRB-9Arg-ScFvJ591), was expressed in E. coli. and purified usingunfolding and refolding steps (FIG. 2B) as described in Example 1. Thebinding of the purified protein to dsRNA was evaluated. dsRB-SCP wasincubated with dsRNA of defined length (500 bp) and the mixture waselectrophoresed on an agarose gel (FIG. 2C). The naked dsRNA control ranat the expected position in the gel (FIG. 2C). The electrophoresis ofdsRNA that had been incubated with dsRB-SCP was retarded in adose-dependent manner (FIG. 2C), confirming that the chimeric proteinbound the dsRNA.

dsRB-SCP Complexed with polyIC Selectively Kills PSMA Over-ExpressingCells by Inducing Apoptosis

We evaluated the killing effect of the dsRB-SCP/polyIC complex usingfour cell lines: LNCaP and VCaP, which over-express PSMA, and MCF7 andPC3, which do not express PSMA. dsRB-SCP selectively delivered polyICinto the PSMA-over-expressing cells (LNCaP and VCaP), killing up to 80%of the cells (FIG. 3A). Cells which do not express PSMA (MCF7 and PC3),were not killed by the treatment (FIG. 3A). The remaining 20% of LNCaPcells were deemed permanently arrested, as no regrowth was observed 250hr after washing out the chimera (350 hr after treatment) (FIG. 3B).dsRB-SCP/polyIC induced cell death by activating apoptotic pathways, asindicated by the cleavage of caspase-3 and PARP (FIG. 3C). In cellstreated with polyIC alone no cleavage of caspase-3 or of PARP wasdetected (FIG. 3C).

dsRB-SCP/polyIC Treatment Induces Cytokine Secretion and Chemotaxis ofImmune Cells

The presence of dsRNA inside the cell activates the production ofanti-proliferative and pro-apoptotic cytokines and chemokines (20). Todetermine whether dsRB-SCP/polyIC can trigger similar effects weanalyzed the production of three main cytokines in the cell: IP-10 andRANTES, both involved in the chemo-attraction of immune cells and IFN-0,which plays a key role in the differentiation of immune cells (21). Thesecretion of IP-10 and RANTES into the medium, as measured by ELISA, waspartially induced by polyIC alone, as reported previously (22).Treatment with dsRB-SCP/polyIC led to a further 2-fold increase in IP-10and RANTES secretion (FIG. 4A-B). IFN-β expression was not affected bypolyIC or dsRB-SCP alone, but treatment with dsRB-SCP/polyIC led to verystrong induction of IFN-β expression, as measured by qRT-PCR (FIG. 4C).

To study whether the secreted cytokines attract immune cells, weexamined whether the medium from dsRB-SCP/polyIC-treated LNCaP cellsinduced the chemotaxis of freshly isolated PBMCs. FIG. 4D shows that anincreased number of PBMCs migrated towards conditioned medium from cellsthat were treated with dsRB-SCP/polyIC compared to medium from untreatedcells.

Bystander Effects Induced by dsRB-SCP/polyIC

We next tested whether the recruited immune cells could evoke animmune-cell-mediated bystander effect. We treated LNCaP-Luc/GFP cells,which stably express luciferase, with a low dose of dsRB-SCP/polyIC,followed by co-incubation with PBMCs. We used luciferase activity as ameasure for the survival of the LNCaP-Luc/GFP cells. Results showederadication of the LNCaP-Luc/GFP cells (FIG. 5A). In contrast, in theabsence of PBMCs, luciferase level was barely affected. These resultssuggest that dsRB-SCP/polyIC induces a powerful immune-cell-mediatedbystander effect.

To evaluate whether dsRB-SCP/polyIC also induces a direct bystandereffect, LNCaP cells were co-incubated with PC3-Luc/GFP cells, which donot express PSMA. dsRB-SCP/polyIC treatment resulted in the killing ofup to 60% of the PC3-Luc/GFP cells (FIG. 5B). Since PC3-Luc/GFP cellsare not targeted by dsRB-SCP/polyIC (FIG. 5B), we infer that the deathof these cells is a result of a direct bystander effect elicited by thedsRB-SCP/polyIC-targeted LNCaP cells. Addition of human PBMCs to thisco-culture system led to a significant increase in the killing rate ofthe PC3-Luc/GFP cells (FIG. 5B), indicating the additional involvementof an immune-cell-mediated bystander effect under these conditions.

dsRB-SCP/polyIC Destroys Tumor Spheroids

We next evaluated the efficacy of dsRB-SCP/polyIC in a 3D tumor spheroidmodel. In vitro 3D models closely resemble the architecture of humantumors (23) and feature high-resistance to anti-cancer drugs (24). LNCaPspheroids were generated and allowed to reach a diameter of 300-400 μm.The spheroids were then transferred to a polyHEMA plate and treatedrepeatedly with dsRB-SCP/polyIC (400 nM dsRB-SCP, 2.5 μg/ml polyIC) overthe course of 5 days. By day 5, the spheroids that were treated withdsRB-SCP/polyIC began to shrink and shed dead cells, while the untreatedspheroids increased in size (FIG. 6A, C). On day 15, the spheroids werestained with calcein AM and propidium iodide to monitor viability (FIG.6A, C). The dsRB-SCP/polyIC-treated spheroids demonstrated significantstructural damage and contained large numbers of dead cells (FIG. 6A,C). In contrast, the untreated spheroids and spheroids treated with onlypolyIC or only dsRB-SCP, maintained a typical intact structure (11),where the cells at the surface were alive and the cells at the core werenecrotic (FIG. 6A, C).

To more closely mimic in vivo conditions and test theimmune-cell-mediated bystander effect on the spheroids, we added PBMCsto treated spheroids. LNCaP-Luc/GFP spheroids were treated once withdsRB-SCP/polyIC, and 24 hr later freshly isolated PBMCs were added tothe culture. Even at the lowest dose of dsRB-SCP/polyIC, spheroiddisassembly was already evident 72 hr after the initiation of thetreatment or 48 hr after PBMCs addition (FIG. 6B). At higher doses,complete spheroid destruction was observed 96 hr after the initiation ofthe treatment. After additional 72 hr, only dead cells were evident withno GFP fluorescence (FIG. 6B). As a control, the same treatment wasperformed in absence of PBMCs. At the end point (168 hr), the treatmentresulted in visible cell death and disassembly of the spheroid (FIG. 6Blower panel) but the effect was weaker compared to the levels observedin the presence of PBMCs. Thus, dsRB-SCP/polyIC has a potent effect onspheroids, and this effect is greatly magnified by the addition ofimmune cells.

Example 3 3.1 Materials and Methods

Cell Culture

A431 cells were grown in DMEM supplemented with 10% fetal calf serum(FCS), penicillin and streptomycin. MDA-MB-468 and MCF7 cells were grownin RPMI 1640 medium supplemented with 10% FCS, penicillin andstreptomycin.

Cloning

The chimeric gene encoding dsRBEC was constructed as follows: The dsRBDof hPKR (nucleotides 558-1057, NM 0027593) was fused to the nucleic acidsequence of hEGF (GenBank: M11936.1). The 3′ sequence of the dsRBD(nucleotides 1028-1057) was changed to GGCCAAACTGGCCTATCTGCAGATCTTATC(SEQ ID NO:24) to optimize codon usage and to introduce new restrictionsites, and a linker (GGCGTGTTCGGGATCCGCCGGCAACCGTGTCCGTCGGAGCGTGGGCAGCTCGAATGGA) (SEQ ID NO:25), encoding ACSGSA CSGSAGNRVRRSVGSSNG (SEQ ID NO:26), was introduced before the hEGFmoiety. The chimera was cloned into the bacterial expression vectorpET28a (Novagen), between restriction sites Ndel and HindIII, thusinserting a Hexa His tag at the N-terminus of the chimera.

dsRBEC Expression

E coli BL21(DE3)/CodonPlus RIL (Stratagene) carrying thepET28a-His6-dsRBEC plasmid was grown in 2xYT [45] supplemented with 1%glucose, 25 μg/ml chloramphenicol, and 30 μg/ml kanamycin at 37° C. toOD600˜0.6. At this point, the bacteria were moved to 23° C. Proteinexpression was induced by adding 0.5 mMIsopropyl-B-D-thiogalactopyranoside (IPTG), and the culture wasincubated at 23° C. for 6 hours longer. The bacterial culture was thencentrifuged at 5000×g for 10 minutes and the pellet was stored at −80°C. until further applications.

Small Scale Purification and RNA Contamination Analysis

The pellet from 10 ml bacterial culture was resuspended in 1 ml lysisbuffer (20 mM Hepes pH 7.5, 0.5M NaCl, 10% glycerol, 10 mM imidazole)and disrupted using a LV1 microfluidizer (Microfluidics). Following 15minutes centrifugation at 15,000×g and 4° C., the cleared supernatantwas loaded onto 50 μl equilibrated Ni Sepharose High Performance beads(GE Healthcare Life Sciences) and rotated for 1 hour at 4° C. Followingtwo washes with lysis buffer, the bound protein was eluted with 2004elution buffer (20 mM Hepes pH 7.5, 0.5M NaCl, 10% glycerol, 500 mMimidazole). Samples from each step (total lysate, soluble fraction,unbound fraction and eluate) were subjected to SDS-PAGE (15%polyacrylamide). The gel was stained with InstantBlue Coomassie basedgel stain (Expedeon) or transferred to nitrocellulose membranes forwestern analysis using anti-His tag antibody (LifeTein, #LT0426, 1:1000dilution). To visualize nucleic acid contamination of the protein, 30 μlof the eluted protein were electrophoresed on a 1% agarose gel. Whererelevant, the protein was treated with RNase A (10 ug/ml) for 30 minutesat 37° C. prior to agarose gel electrophoresis. The gel was stained withethidium bromide following electrophoresis. For purification underdenaturing conditions, the bacterial pellet was resuspended with lysisbuffer containing 4M urea, and was incubated at 4° C. for 1.5 hoursprior to centrifugation.

On-Column Purification and Renaturation

The pellet from 500 ml of bacterial culture was resuspended with 40 mllysis buffer supplemented with 4M urea and disrupted using a LV1microfluidizer. The lysate was incubated at 4° C. for 1.5 hours, andcleared by centrifugation for 30 minutes at 15,000×g at 4° C. The clearsupernatant was loaded onto 4 ml equilibrated Ni Sepharose beads andincubated for an additional hour at 4° C. in a 50 ml tube. The beadswere then loaded onto a 4 ml C 10/10 column (GE Healthcare) andconnected to an AKTA Explorer system (GE Healthcare). The protein wasrefolded by gradually reducing the concentration of urea. A gradientprogram was used with Buffer A (20 mM Hepes pH 7.5, 0.5M NaCl, 10 mMimidazole, 10% glycerol, 4M urea) and Buffer B (20 mM Hepes pH 7.5, 0.5MNaCl, 10 mM imidazole, 10% glycerol). The gradient was programmed toreach 100% B in 30 column volumes (CV) at 0.2 ml/minute flow. The columnwas washed with 4 CV of buffer (20 mM Hepes pH 7.5, 0.5M NaCl, 10%glycerol) containing 25 mM imidazole and with another 4 CV of bufferwith 50 mM imidazole. The chimera was eluted in the same buffer, towhich imidazole had been added to 500 mM. The protein eluted from the NiSepharose column was loaded onto a 320 ml column of Superdex 75 (GEHealthcare Life Sciences), which had been pre-equilibrated with buffercontaining: 20 mM Hepes pH 7.4, 10% glycerol, 500 mM NaCl, for gelfiltration. The purified protein was divided into aliquots and stored at−80° C.

PolyIC Electrophoretic Mobility Shift Assay (EMSA)

Low molecular weight (LMW) polyIC (Invitrogen) was labeled with Cy3using the Label I T® nucleic acid labeling kit (Mirus) according to themanufacturer's protocol. 0.5 μg of labeled polyIC was incubated for 30minutes with increasing amounts of purified dsRBEC (0.5-4 μg), followedby electrophoresis of the mixture on a 1.5% TAE-agarose gel. The gel wasvisualized using the MF-ChemiBlS system (DNR Bio-Imaging Systems).

¹²⁵I-EGF Displacement Assay

A431 cells were harvested by trypsinization and resuspended in PBSsupplemented with 1% BSA and plated in 96-well MultiScreen filter plates(Millipore) (5,000 cells per well). Following 30 minutes incubation onice with gentle shaking, the medium was aspirated using aMultiScreen^(HTS) vacuum manifold (Millipore) and replaced with ice-coldPBS using a MultiScreenHT supplemented with 0.1% BSA. Increasingconcentrations (0-16 nM) of the dsRBEC or hEGF (PeproTech) were added tothe wells, in triplicate. Following 30 minutes incubation on ice, thecells were supplemented with ¹²⁵I-EGF (0.1 nM, PerkinElmer) andincubated 4 h longer on ice, with gentle shaking. The medium was thenaspirated, and the cells were washed five times with ice-cold PBSsupplemented with 0.1% BSA and the plate was left under vacuum forcomplete drying. The MultiScreen plate was then exposed to a phosphorimager plate (BAS-IP MS 2040 Fuji Photo Film) for 72 hours. An ¹²⁵I-EGFcalibration curve (0-10 fmol) was used to convert pixels into absoluteconcentrations. The plate was scanned using a FujiFilm Fluorescent ImageAnalyzer FLA-3000. Nonlinear regression (competitive binding one siteanalysis) was performed on the data using GraphPad Prism™, Version 5.0.Kd values were calculated as the means from three independent bindingexperiments, each of which comprised triplicate samples. The Kd of¹²⁵I-EGF, which was required for the analysis of the displacement data,was measured as previously described by Abourbeh et al [46] (FIG. 8).

EGFR Phosphorylation

MDA-MB-468 cells were plated in 6-well plates (500,000 cells per well)in RPMI 1640 medium and 10% FCS. 24 hours after plating, the cells werewashed twice with PBS and the medium was replaced with RPMI mediumlacking FCS. 16 hours later, the cells were treated with dsRBEC avarious concentrations for 15 minutes. The cells were then harvestedwith hot Laemmli sample buffer. EGFR phosphorylation was evaluated bywestern blot analysis, using anti-phospho (Y1068)-EGFR antibody (CellSignaling Technology, #2234, 1:1000 dilution), anti-EGFR antibody (SantaCruz, sc-03, 1:1000 dilution) and anti-GAPDH antibody (Santa Cruz,sc-25778, 1:2000 dilution).

Confocal Microscopy

MDA-MB-468 (10000 cells/well) and MCF7 (7000 cells/well) cells wereplated in a u-Slide 8 Well Glass Bottom plate (Ibidi). 48 hours afterplating, the medium was replaced with fresh medium containing 1 μMsulforhodamine G (Biotium Inc.) and the plate was placed in the 37° C.chamber of a confocal microscope (FV-1200 Olympus, Japan). The cellswere then treated with 1 μg/ml Cy3-polyIC alone, or 1 μg/ml Cy3-polyICwhich had been pre-incubated with dsRBEC (polyIC:dsRBEC, weight:weightratio of 1:2). PolyIC internalization was monitored for 2 hours. Forendosomal localization we treated MDA-MB-468 cells with polyIC/dsRBEC(as described above) and 5 μg/ml AlexaFluor 647-labeled transferrin(Jackson ImmunoResearch Laboratories, Inc) simultaneously.

EGFR Level

The expression of EGFR in MDA-MB-468 and MCF7 cells was analyzed bywestern blot using anti-EGFR antibody (Santa Cruz, sc-03, 1:1000dilution). Anti-GAPDH antibody (Santa Cruz, sc-25778, 1:2000 dilution)was used a loading control. Expression of surface EGFR in the two celllines was assessed by flow cytometry using PE-conjugated anti-human EGFRantibody (BioLegend, #352903).

Survival Assay

MDA-MB-468 and MCF7 cells were plated in 96-well plates (5000 and 2000cells per well, respectively). The next day the medium was refreshed andthe cells were treated with polyIC, dsRBEC or polyIC which had beenpre-incubated with dsRBEC (polyIC:dsRBEC weight:weight ratio of 1:2). 72hours following the treatment, the survival of the cells was measured bythe methylene blue colorimetric assay [47].

Apoptosis

FACS

MDA-MB-468 and MCF7 cells were plated in a 24-well plate (150,000 and100,000 cells per well, respectively). The following day the cells weretreated with polyIC (1 μg/ml), dsRBEC (2 μg/ml) or polyIC (1 μg/ml)which had been pre-incubated with dsRBEC (polyIC:dsRBEC weight:weightratio of 1:2) for 8 hours. Annexin V/Propidium iodide (PI) staining wasperformed using the MBL MEBCYTO apoptosis kit according to themanufacturer's guidelines and analyzed using flow cytometry, BD FACSARIAIII (BD Biosciences, USA).

Caspase-3 and PARP Cleavage

MDA-MB-468 cells were plated in 12-well plates (250,000 cells per well).The next day the medium was refreshed and the cells were treated withpolyIC (1 μg/ml), dsRBEC (2 μg/ml) or polyIC (1 μg/ml) which had beenpre-incubated with dsRBEC (polyIC:dsRBEC weight:weight ratio of 1:2). 4hours following the treatment, the cells were harvested with hot Laemmlisample buffer and subjected to SDS-PAGE (12% polyacrylamide) and westernblotting, using anti-Cleaved Caspase-3 antibody (Cell SignalingTechnology, #9661, 1:1000 dilution), anti-PARP antibody (Cell SignalingTechnology, #9542, 1:1000 dilution) and anti-GAPDH antibody (Santa Cruz,sc-25 778, 1:2000 dilution).

RNA Isolation and Reverse Transcriptase-Real Time Polymerase ChainReaction (qRT-PCR)

MDA-MB-468 cells were treated with polyIC (1 μg/ml), dsRBEC (2 μg/ml) orpolyIC (1 μg/ml) which had been pre-incubated with dsRBEC (polyIC:dsRBECweight:weight ratio of 1:2) for 2 and 4 hours. Total RNA was isolatedfrom MDA-MB-468 cell using the EZ-10 DNA away RNA-Mini-prep Kit (BioBasic) according to the manufacturer's instructions. 1 μg of total RNAwas reverse-transcribed using the High-Capacity cDNA ReverseTranscription Kit (Applied Biosystems) and the resulting cDNA was usedfor qRT-PCR analysis (Fast SYBR Green; Applied Biosystems) using theprimer pairs listed in Table 2.

Gene expression was normalized to GAPDH gene expression and compared tosamples from vehicle-treated cells. Fold change was quantified using the^(ΔΔ)CT method.

TABLE 2 qRT-PCR primer sequences Gene Primer sequence GAPDH F:5′ GAGCCACATCGCTCAGAC 3′ (SEQ ID NO: 27) R: 5′ CTTCTCATGGTTCACACCC 3′(SEQ ID NO: 28) IFN-beta F: 5′ ATGACCAACAAGTGTCTCCTCC 3′ (SEQ ID NO: 29)R: 5′ GCTCATGGAAAGAGCTGTAGTG 3′ (SEQ ID NO: 30) CCL5 F:5′ CGCTGTCATCCTCATTGCTACTG 3′ (SEQ ID NO: 31) R:5′ GCAGGGTGTGGTGTCCGAG 3′ (SEQ ID NO: 32) IP-10 F:5′ GCCAATTTTGTCCACGTGTTG 3′ (SEQ ID NO: 33) R:5′ AGCCTCTGTGTGGTCCATCCT 3′ (SEQ ID NO: 34) TNF-alpha F:5′ GTGCTTGTTCCTCAGCCTCTT 3′ (SEQ ID NO: 35) R:5′ GGCCAGAGGGCTGATTAGAGAG 3′ (SEQ ID NO: 36)

ELISA

MDA-MB-468 and MCF 7 cells were plated in 96-well plates (10,000 and7,000 cells per well, respectively). The next day the medium wasrefreshed and the cells were treated with polyIC, dsRBEC or polyIC whichhad been pre-incubated with dsRBEC (polyIC:dsRBEC weight:weight ratio of1:2). 24 hours following the treatment, the medium was collected.Interferon gamma-induced protein 10 (IP-10), chemokine (C-C motif)ligand 5 (CCLS) and tumor necrosis factor alpha (TNF-alpha) proteinswere quantified using ABTS ELISA Development Kits (PeproTech) accordingto the manufacturer's protocol. Interferon beta (IFN-beta) protein wasquantified using a bioluminescent ELISA kit (LumiKine) according to themanufacturer's protocol.

Statistical Analysis

GraphPad Prism was used for all statistical analysis. One-way ANOVA andTukey post-test were used to analyze the ELISA experiments. The survivalassay was analyzed using two-way ANOVA and Bonferroni post-testanalysis.

3.2 Results

Expression and Purification of dsRBEC

We designed a chimeric protein vector, dsRBEC, for the targeted deliveryof polyIC to EGFR over-expressing tumor cells. This vector comprised thedsRBD of human PKR fused via a linker to human EGF (see Materials andMethods and FIG. 7).

dsRBEC was in E. coli efficiently expressed as a His6-tagged proteinBL21(DE3)/CodonPlus RIL. As a first step, the chimera was purified usingNi Sepharose High Capacity resin. Under native conditions the yield ofprotein was very low, due to poor solubility and poor binding to theresin (FIG. 9A). Furthermore, the purified dsRBEC was contaminated withnucleic acids, as indicated by the high OD₂₆₀/OD₂₃₀ ratio of 1.92 and byethidium bromide staining. When we treated the eluted protein with RNaseA prior to electrophoresis, ethidium bromide staining was no longerdetectable (FIG. 9B). This indicated that the contaminating nucleic acidwas host RNA bound to dsRBEC, presumably at its dsRBD.

In order to remove the contaminating RNA under native conditions, wetook several approaches, including treatment with RNase A orpolyethyleneimine (PEI). However, these treatments resulted inprecipitation of our protein. We therefore attempted to purify thechimera under denaturing conditions. We lysed the bacteria and bound thelysates to Ni Sepharose beads in the presence of 4M urea. Under theseconditions, the protein was highly soluble and bound the column withincreased affinity (FIG. 9C). The amount of contaminating RNA in theeluate was significantly reduced, as detected by the decrease inOD₂₆₀/OD₂₃₀ ratio to 0.7 and by the lack of staining with ethidiumbromide (FIG. 9D). Thus, denaturing conditions facilitated the removalof the contaminating RNA, improved the solubility of the chimera andincreased its yield.

The next step was to scale up the purification procedure. To processlarger amounts of lysate, we decided to use the AKTA Explorer system.This system made it very easy to perform on-column refolding, with acontinuous, gradual reduction in urea concentration, while the proteinwas still bound to the Ni Sepharose resin. The immobilization of aprotein on a column prevents protein aggregation caused byintermolecular interactions [48-51]. To remove remaining proteinimpurities (FIG. 9E, Lane 2), the eluate from the Ni Sepharose columnwas subjected to a second purification step, Superdex75 gel filtration(FIG. 9E, Lane 3). The total yield of purified protein was 6 mg from 0.5L of bacterial culture.

dsRBEC Binds polyIC

To evaluate the ability of dsRBEC to bind polyIC, we performed an EMSAtest. Incubation of dsRBEC with Cy3-labeled polyIC (Cy3-polyIC) retardedthe migration of Cy3-polyIC on a 2% agarose gel in a dose-dependentmanner (FIG. 11A), showing that the dsRBD moiety of dsRBEC is competentto bind polyIC.

dsRBEC Binds EGFR with High Affinity

The affinity of the purified dsRBEC to EGFR was measured by competitivebinding experiments, using radio-labeled EGF. We measured the decreasein¹²⁵I-EGF binding in the presence of increasing concentrations ofunlabeled hEGF or of dsRBEC (FIG. 11B). Kd values of 0.54±0.18 nM forhEGF and 3.38±0.75 nM for dsRBEC were calculated using competitivebinding one site analysis (GraphPad Prism 5). dsRBEC retains highaffinity to EGFR, suggesting that the N-terminal fusion to the dsRBDmoiety did not detract from the affinity of the EGF moiety for EGFR, andthat the refolding protocol regenerated the active conformation.

EGF binding to EGFR leads to receptor autophosphorylation, followed byclathrin-coated pit mediated endocytosis [52]. To test for productivebinding of dsRBEC to EGFR, we measured the autophosphorylation oftyrosine residue 1068 of EGFR, a characteristic EGFR autophosphorylationsite, which is critical for EGFR internalization [52]. Since A431 cellshave higher levels of basal EGFR phosphorylation [53], we usedMDA-MB-468 cells, which also express high levels of EGFR, but show muchless basal phosphorylation [54]. dsRBEC treatment for 15 minutes inducedthe phosphorylation of tyrosine 1068 in a dose-dependent manner (FIG.11C). This indicates that dsRBEC binds EGFR correctly and can inducereceptor phosphorylation, which is necessary for internalization.

dsRBEC Selectively Induces polyIC Internalization in EGFROver-Expressing Cells

Next, we tested whether dsRBEC could deliver polyIC selectively intoEGFR over-expressing cells. We compared MDA-MB-468 cells, whichover-express EGFR, and MCF7 cells, which express low or undetectablelevels of the receptor [55] (FIG. 12A, D). Using confocal microscopy wedemonstrated that Cy3-polyIC complexed with dsRBEC (Cy3-polyIC/dsRBEC)was internalized into MDA-MB-468 cells, whereas no internalization wasobserved in MCF7 cells (FIG. 12B). Naked Cy3-polyIC was not internalizedby either cell line (FIG. 12B). The punctate fluorescent pattern ofCy3-polyIC when carried by dsRBEC is indicative of endosomal entrapment[56-58]. To verify endosomal localization we treated MDA-MB-468 cellswith Cy3-polyIC/dsRBEC in the presence of AlexaFluor647-conjugatedtransferrin, a recycling endosomal marker. Indeed, we observed strongco-localization of Cy3-polyIC with transferrin (FIG. 12C). These datacorroborate our findings that the dual functional chimera, dsRBEC, canbind both EGFR and polyIC. Thus, dsRBEC induces targeted, selectivedelivery of polyIC into EGFR over-expressing cells, where it accumulatesin endosomes.

Targeted Delivery of polyIC by dsRBEC Leads to Apoptosis ofMDA-MB-468Cells

We next evaluated the survival of MDA-MB-468 cells, following treatmentwith polyIC/dsRBEC. PolyIC/dsRBEC led to reduced survival of MDA-MB-468cells, in a dose-dependent manner, whereas MCF7 cells were unaffected bythe treatment even at the highest concentration tested (1 μg/ml) (FIG.13A). A complex of 0.5 μg/ml polyIC with 1 μg/ml dsRBEC led to a 90%decrease in survival of MDA-MB-468 cells (FIG. 13A). The chimera dsRBECalone had a slight inhibitory effect on MDA-MB-468 cell survival, whichis consistent with earlier reports that EGF alone can mediate apoptosisof this cell line [59, 60]. Notably, naked polyIC had a much weakereffect on cell survival than polyIC/dsRBEC (***, P<0.0001 for effect ofpolyIC/dsRBEC vs polyIC alone, for polyIC/dsRBEC vs dsRBEC alone and forpolyIC/dsRBEC vs vehicle at all tested concentrations).

We next tested whether the decreased cell survival upon treatment withPolyIC/dsRBEC was due to apoptosis. In MDA-MB-468 cells treated withpolyIC/dsRBEC, cleavage of both PARP and caspase-3 was evident as soonas 4 hours after treatment initiation (FIG. 13B). Furthermore, there wasa strong increase in Annexin V-positive cells after 8 hours of treatmentwith polyIC/dsRBEC (FIG. 13C). In contrast, treatment with naked polyICor dsRBEC alone had no or little effect on PARP or caspase-3 cleavage(FIG. 13B) or on Annexin V staining (FIG. 13C). MCF7 cells were notaffected by the treatment and did not show any sign of apoptosis (FIG.13C). These results indicate that polyIC/dsRBEC, but not polyIC alone,induces selective apoptosis of EGFR-over-expressing cells.

PolyIC Delivery by dsRBEC Induced the Expression of Pro-InflammatoryCytokines from MDA MB-468 Cells

PolyIC has been reported to stimulate the secretion of pro-inflammatorycytokines [31-33]. We therefore measured the effect of polyIC/dsRBECtreatment on pro-inflammatory cytokine production. MDA-MB-468 cells weretreated with polyIC/dsRBEC for 2 or 4 hours, and the mRNAs of IFN-beta,IP-10, CCLS and TNF-alpha were measured using qRT-PCR (FIG. 14A-D).Treatment with polyIC/dsRBEC led to profound induction of cytokine mRNAexpression. In contrast, treatment with naked polyIC led tosubstantially less mRNA expression. Treatment with dsRBEC alone did notaffect the expression of these cytokines.

To verify that polyIC/dsRBEC induces increased cytokine secretion, wemeasured the protein levels of these cytokines using ELISA. After 24hours of treatment with polyIC/dsRBEC, significant levels of the fourcytokines were detected in the medium (FIG. 14E-H). At the highestconcentration of polyIC/dsRBEC tested (1 μg/ml polyIC, 2 μg/ml dsRBEC)TNF-alpha could not be detected, probably because the cells underwentapoptosis before they were able to secrete TNF-alpha. Interestingly,although naked polyIC induced the transcription of all four tested genes(FIG. 14A-D), only IP-10 was detected by ELISA, at substantially lowerlevels than were induced by polyIC/dsRBEC. In addition, none of thetested cytokines were detected in the medium of MDA-MB-468 cells treatedwith dsRBEC alone (FIG. 14E-H) or in the medium of MCF7 cells in allsets of treatments (data not shown). Thus, polyIC/dsRBEC induced boththe mRNA transcription and protein secretion of pro-inflammatorycytokines in a highly specific manner.

Taken together, our data show that dsRBEC can be efficiently purifiedfrom E. coli in an active conformation. dsRBEC is an efficient vectorfor targeting polyIC into EGFR over-expressing tumor cells, leading toapoptosis and cytokine secretion.

3.3 Discussion

In the present study we present the development of a novel, recombinantprotein carrier, dsRBEC, to selectively deliver polyIC into EGFRover-expressing tumors. dsRBEC is a bifunctional protein, with a dsRBDto bind poll and an EGF domain to deliver the polyIC into EGFRover-expressing cells. dsRBEC effectively and selectively induced polyICinternalization into EGFR over-expressing cells, inducing cell death andcytokine secretion. As opposed to current anti-EGFR therapies, whichinhibit EGFR activity per se, we exploit EGFR expression and activationas the Achilles' heel of the tumor. We use the over expression of thereceptor as a selective entryway into the cell.

Upon purification from E. coli, RNA binding proteins are frequentlycontaminated by non-specific host nucleic acids, thus complicating theprotein purification procedure [61, 62]. One widely used method toremove nucleic acid contaminants is precipitation with PEI [62-64]. Thismethod, however, is time-consuming and requires the titration of PEIconcentration and of the ionic strength [62, 65]. In addition, traces ofPEI can interfere with the function of the purified protein [64].Furthermore this method is also difficult to reproduce [62]. RNase andDNase can be used for removal of nucleic acid contamination, but anytraces of the nucleases must be removed [61, 66]. Applying these methodsto our protein resulted in precipitation of dsRBEC.

Marenchino et al. succeeded in purifying His-tagged HIV-1 Rev bydestabilizing the protein-RNA interactions using 8M urea, withsubsequent on-column refolding [62]. We found that 4M urea wassufficient for the complete removal of contaminating RNA from dsRBEC.Denaturation using low concentrations of urea preserves the native-likeprotein structure, minimizing the aggregation of protein moleculesduring refolding, and facilitating recovery of the protein's biologicalactivity [67]. Furthermore, the lower concentration of urea in our studyallowed us to purify the protein at 4° C. This would have beenimpossible using 8M urea, which crystallizes at cold temperatures. Wedesigned a simple, reproducible on-column refolding procedure, which waseasily scaled up using the AKTA Explorer system. This straightforwardprocedure can be used for other RNA binding proteins. The use of urea toremove RNA contamination also increased dramatically the yield of thepurified protein, since it improved the solubility and the binding tonickel resin. Following purification, we confirmed that both domains ofthe bifunctional chimeric protein were active, by showing that it couldbind polyIC efficiently, as well as bind to and activate the EGFR.

In this study, we demonstrate that dsRBEC induces the uptake of polyIConly in cells that over-express the EGFR. Since healthy cells expressEGFR at low levels, we expect this treatment to be highly selective, andnot to affect non-cancerous cells. Furthermore, naked polyIC has littleor no effect on tumor cells at the concentrations that we used. Previousstudies have shown the potency of naked polyIC to induce tumor cellapoptosis through the TLR3 pathway. However much higher concentrations(25-50 μg/ml) of polyIC were used in these studies [27, 28, 33].Although TLR3 is expressed on both the cell surface and the endosomalmembrane, it is believed to bind its ligand and undergo proteolyticactivation in the endosomal compartment [68-71]. Therefore, wehypothesize that the reason polyIC/dsRBEC is much more potent than nakedpolyIC, is because polyIC/dsRBEC accumulates in the endosome, where itstrongly activates TLR3. Moreover, it has been previously reported thatEGFR is essential for TLR3 activation [72], therefore, activation of thereceptor by dsRBEC may contribute to the increased potency of polyIC. Inaddition, we cannot completely exclude the possibility that some of ourtargeted polyIC reaches the cytoplasm. In the cytoplasm, polyIC canactivate other dsRNA-binding proteins, such as PKR, retinoicacid-inducible gene I (RIG-1), and melanoma differentiation-associatedgene 5 (MDAS) [73-75], which may contribute to the pro-apoptotic effectcaused by the internalized polyIC. Our delivery system could advance theclinical use of polyIC, by allowing the use of lower doses and reducingtoxicity. We demonstrated that polyIC/dsRBEC strongly induces theexpression of IFN-beta, IP-10, CCLS and TNF-alpha. These cytokinesshould provide a second line of therapeutic effect, activating andrecruiting immune cells to the site of the tumor. Type I IFNs havepreviously been shown to regulate the activation of innate immune cells,including macrophages, natural killer cells and APCs. Type I IFNsregulate the activation and proliferation of adaptive immune cells,directly via the IFN receptor and indirectly by activation of APCs or byup-regulation of MHC and co-stimulatory molecules [76, 77]. Type I IFNscan also directly affect cancer cells, by promoting cell cycle arrestand apoptosis [78, 79]. Thus, the cytokines produced in response totumor-targeted polyIC can induce a “bystander” effect, restoringantitumor immune surveillance and eradicating neighboring tumor cellsthat do not over-express the receptor. Since most tumors areheterogenic, this bystander effect is extremely beneficial.

In conclusion, we have developed a bi-domain recombinant chimericprotein that is capable both of binding polyIC and of delivering it intoEGFR over-expressing tumors, inducing tumor cell death. On the basis ofthis study, we suggest that dsRBEC-delivered polyIC may be a promisingtherapy for EGFR over-expressing tumors. We have created a platformtechnology, with dsRBEC as the prototype recombinant protein. Byreplacing the EGF moiety with targeting moieties for differentreceptors, this approach can be applied to additional tumor types.

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In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

The following items should be read as preferred embodiments:

-   1. A recombinant protein comprising a double stranded RNA (dsRNA)    binding domain and a target-binding moiety.-   2. The recombinant protein of embodiment 1, wherein said dsRNA    binding domain and said target-binding moiety are connected by a    spacer peptide.-   3. The recombinant protein of embodiment 1 or 2, further comprising    a cytolytic peptide.-   4. The recombinant protein of any one of embodiments 1 to 3, wherein    said dsRNA binding domain comprises one or more double-stranded    RNA-binding motif (dsRBM), i.e. an alpha-beta-beta-beta-alpha fold.-   5. The recombinant protein of embodiment 4, wherein said one or more    dsRBM is selected from a dsRBM of dsRNA dependent protein kinase    (PKR), TRBP, PACT, Staufen, NFAR1, NFARZ, SPNR, RHA or NREBP.-   6. The recombinant protein of embodiment 5, wherein said dsRNA    binding domain comprises two dsRBMs of a PKR, optionally connected    by a flexible linker.-   7. The recombinant protein of embodiment 6, wherein said dsRNA    binding domain is selected from the dsRNA binding domain of human    PKR and said two dsRBMs are connected by a flexible linker; or the    full length human PKR.-   8. The recombinant protein of embodiment 7, wherein said dsRNA    binding domain comprises amino acid residues 1-197 of human PKR.-   9. The recombinant protein any one of embodiments 1 to 3, wherein    said target-binding moiety comprises (i) a ligand to a cell surface    receptor; (ii) an antibody, such as a humanized antibody; a human    antibody; a functional fragment of an antibody; a single-domain    antibody, such as a Nanobody; a recombinant antibody; and a single    chain variable fragment (scFv); (ii) an antibody mimetic, such as an    affibody molecule; an affilin; an affimer; an affitin; an alphabody;    an anticalin; an avimer; a DARPin; a fynomer; a Kunitz domain    peptide; and a monobody; or (iii) an aptamer.-   10. The recombinant protein of embodiment 9, wherein said    target-binding moiety binds a tumor-associated antigen.-   11. The recombinant protein of embodiment 10, wherein said    tumor-associated antigen is selected from epidermal growth factor    receptor (EGFR), human epidermal growth factor receptor 2 (HER2),    prostate surface membrane antigen (PSMA), fibroblast growth factor    receptor (FGFR), colony stimulating factor 1 receptor (CSF-1R),    platelet-derived growth factor receptors (PDGFR), insulin-like    growth factor 1 receptor (IGF-1R) and MET.-   12. The recombinant protein of embodiment 11, wherein said    target-binding moiety is an EGFR ligand, such as an EGF or the    peptide GE11 of the sequence YHWYGYTPQNVI (SEQ ID NO:22); an    anti-EGFR antibody, such as an anti-EGFR scFv or a humanized or    human anti-EGFR antibody; or an anti-EGFR affibody.-   13. The recombinant protein of embodiment 12, wherein said EGF is    human EGF and said EGFR is human EGFR.-   14. The recombinant protein of embodiment 11, wherein said    target-binding moiety is a human anti-epidermal growth factor    receptor 2 (HER2) antibody, such as an anti-HER2 scFv or a humanized    or human anti-HER2 antibody; or an anti-HER2 affibody.-   15. The recombinant protein of embodiment 11, wherein said    target-binding moiety is a prostate surface membrane antigen (PSMA)    ligand, such as DUPA or an analog thereof an anti-PSMA antibody,    such as an anti-PSMA scFv or a humanized or human anti-PSMA antibody    (e. g. the full length antibody J591); or an anti-PSMA affibody.-   16. The recombinant protein of embodiment 15, wherein said PSMA is    human PSMA.-   17. The recombinant protein of any one of embodiments 1 to 3,    wherein said spacer peptide is selected from an oligopeptide    comprising a protease recognition sequence; a homo-oligopeptide of a    positively charged amino acid (at physiological pH), such as    arginine; a peptide of the sequence ACSGSACSGSAGNRVRRSVGSSNG (SEQ ID    NO:23) or a homolog thereof a cytolytic peptide; or a combination    thereof-   18. The recombinant protein of embodiment 17, wherein said    homo-oligopeptide of arginine is Arg8.-   19. The recombinant protein of any one of embodiments 1 to 3 or 17,    wherein said cytolytic peptide is selected from Melittin or    Candidalysin.-   20. The recombinant protein of embodiment 19, wherein said cytolytic    peptide is positioned within the spacer peptide or within the    N-terminus of the recombinant protein.-   21. The recombinant protein of any one of embodiments 1 to 3,    comprising the dsRNA binding domain of human PKR wherein said two    dsRBMs are connected by a flexible linker; and a target-binding    moiety selected from an anti-human EGFR antibody, an anti-human HER2    antibody and an anti-PSMA antibody, wherein said dsRNA binding    domain and said target-binding moiety are connected by an Arg8    spacer peptide.-   22. The recombinant protein of embodiment 21, comprising the dsRNA    binding domain of human PKR wherein the two dsRBMs are connected by    a flexible linker; and an anti-human EGFR antibody connected by an    Arg8 peptide.-   23. The recombinant protein of embodiment 21, comprising the dsRNA    binding domain of human PKR wherein the two dsRBMs are connected by    a flexible linker; and an anti-human HER2 antibody connected by an    Arg8 peptide.-   24. The recombinant protein of embodiment 21, comprising the dsRNA    binding domain of human PKR wherein the two dsRBMs are connected by    a flexible linker; and an anti-human PSMA antibody connected by an    Arg8 peptide.-   25. The recombinant protein of any one of embodiments 1 to 24, which    is essentially free of RNA.-   26. A complex comprising the recombinant protein of any one of    embodiments 1 to 24 and dsRNA.-   27. The complex of embodiment 26, wherein said dsRNA is    PKR-activating dsRNA, such as dsRNA comprising a polyinosinic acid    strand and a polycytidylic acid strand (poly 1C).-   28. The complex of embodiment 27, wherein said poly IC comprises at    least 22 ribonucleotides in each strand, for example, 85-300    ribonucleotides in each strand.-   29. The complex of embodiment 26, wherein said dsRNA comprises at    least on siRNA sequence directed against e. g. a pro-oncogenic    protein.-   30. The complex of any one of embodiments 26 to 29, comprising the    dsRNA binding domain of human PKR wherein said two dsRBMs are    connected by a flexible linker; and a target-binding moiety selected    from an anti-human EGFR antibody, an anti-human HER2 antibody and an    anti-PSMA antibody, wherein said dsRNA binding domain and said    target-binding moiety are connected by an Arg8 spacer peptide, and    said poly IC or siRNA is non-covalently associated with said dsRNA    binding domain.-   31. A pharmaceutical composition comprising the complex of any one    of embodiments 26 to 30 and a pharmaceutically acceptable carrier.-   32. A nucleic acid molecule comprising a nucleic acid sequence    encoding the recombinant protein of any one of embodiments 1 to 24.-   33. The nucleic acid molecule of embodiment 32, wherein said    sequence is optimized for expression in a bacterial or plant host    cell, preferably a plant host cell.-   34. A vector comprising at least one control element, such as a    promoter and terminator, operably linked to the nucleic acid    molecule of embodiment 32 or 33, wherein said at least one control    element is optimized for expression in a bacterial or plant host    cell, preferably a plant host cell.-   35. A method for manufacturing a recombinant protein comprising a    dsRNA binding domain and a target-binding moiety, comprising    expressing the nucleic acid molecule of embodiment 32 or 33 or the    vector of embodiment 34 in a bacterial or plant cell and extracting    said recombinant protein from said cells.-   36. The method of embodiment 35, further comprising removing    contaminating host cell RNA from and isolating said recombinant    protein.-   37. The method of embodiment 36, wherein said removing of    contaminating host cell RNA from said recombinant protein comprises    contacting said recombinant protein with urea, e. g. 4M urea, and    refolding said recombinant protein.-   38. The method of any one of embodiments 35 to 37, comprising    expressing the nucleic acid molecule or vector in a plant cell, such    as a tobacco or carrot cell, either in suspension or in a whole    plant.-   39. A method for treatment of cancer characterized by expression of    a tumor-associated antigen, said method comprising systemically    administering to a patient in need the complex of any one of    embodiments 26 to 30 or the pharmaceutical composition of embodiment    31.-   40. The method of embodiment 39, wherein said cancer is selected    from a cancer characterized by EGFR-overexpressing cells, a cancer    characterized by HER2-overexpressing cells and prostate cancer.-   41. The method of embodiment 40, wherein said cancer characterized    by EGFR-overexpressing cells is selected from    non-small-cell-lung-carcinoma, breast cancer, glioblastoma, head and    neck squamous cell carcinoma, colorectal cancer, adenocarcinoma,    ovary cancer, bladder cancer or prostate cancer, and metastases    thereof-   42. The method of embodiment 40, wherein said cancer characterized    by HER2-overexpressing cells is selected from breast cancer, ovarian    cancer, stomach cancer, and aggressive forms of uterine cancer, such    as uterine serous endometrial carcinoma.-   43. The method of embodiment 42, wherein said cancer characterized    by HER2-overexpressing cells is Herceptin/trastuzumab resistant    cancer.-   44. The method of any one of embodiments 40 to 43, wherein said    complex comprises the dsRNA binding domain of human PKR wherein said    two dsRBMs are connected by a flexible linker; and a target-binding    moiety selected from an anti-human EGFR antibody, an anti-human HER2    antibody and an anti-PSMA antibody, wherein said dsRNA binding    domain and said target-binding moiety are connected by an Arg8    spacer peptide, and said polyIC or siRNA is non-covalently    associated with said dsRNA binding domain.-   45. The method of any one of embodiments 40 to 44, further    comprising administering immune cells, such as tumor-infiltrating    T-cells (T-TILs), tumor specific engineered T-cells, or peripheral    blood mononuclear cells (PBMCs).-   46. A chimeric recombinant protein comprising:

a double stranded RNA (dsRNA) binding domain; and

a target binding moiety that binds to prostate surface membrane antigen(PSMA).

-   47. The chimeric recombinant protein of embodiment 46, further    comprising a spacer peptide between the dsRNA binding domain and the    target binding moiety.-   48. The chimeric recombinant protein of embodiment 46 or embodiment    47, wherein the dsRNA binding domain comprises at least one    double-stranded RNA-binding motif (dsRBM).-   49. The chimeric recombinant protein of embodiment 48, wherein the    at least one dsRBM is selected from a dsRBM of dsRNA dependent    protein kinase (PKR), TRBP, PACT, Staufen, NFAR1, NFAR2, SPNR, RHA,    and NREBP.-   50. The chimeric recombinant protein of embodiment 48, wherein the    at least one dsRBM comprises a polypeptide sequence at least 70%    identical to amino acids 1-197 of human PKR as set forth as SEQ ID    NO: 18.-   51. The chimeric recombinant protein of any one of embodiments    46-50, wherein the target binding moiety is a polypeptide, antibody,    antibody fragment, or antibody mimetic.-   52. The chimeric recombinant protein of embodiment 47, wherein the    spacer peptide is selected from the group consisting of an    oligopeptide comprising a protease recognition sequence; a    homo-oligopeptide of a positively charged amino acids; and a    combination thereof-   53. The chimeric recombinant protein of embodiment 52, wherein the    spacer peptide is a homo-oligopeptide of arginine.-   54. The chimeric recombinant protein of any one of embodiments    46-53, wherein the double stranded RNA (dsRNA) binding domain is at    least one dsRNA binding domain of human PKR as set forth in SEQ ID    NO: 18, or a functional variant thereof, wherein the spacer peptide    is ARG9 as set forth in SEQ ID NO: 5, or a functional variant    thereof, and wherein the target binding moiety is a single chain    anti-PSMA antibody as set forth in SEQ ID NO: 20, or a functional    variant thereof-   55. The chimeric recombinant protein of embodiment 54, comprising a    polypeptide at least 70% identical to the sequence set forth as SEQ    ID NO: 3.-   56. A complex comprising the chimeric recombinant protein of any one    of embodiments 46 to 45 and dsRNA.-   57. The complex of embodiment 56, wherein the dsRNA comprises a    polyinosinic acid strand and a polycytidylic acid strand (poly IC).-   58. A nucleic acid comprising a nucleic acid sequence encoding the    recombinant protein of any one of embodiments 46 to 55.-   59. The nucleic acid of embodiment 58, wherein the nucleic acid    sequence is optimized for expression in a bacterial or plant host    cell.-   60. The chimeric recombinant protein of any one of embodiments 46-55    or the complex of embodiment 56 or embodiment 57 for use in    treatment of prostate cancer or inhibition of the development of    tumor neovasculature.-   61. A method for treatment of prostate cancer or inhibition of tumor    neovasculature development comprising, administering to a subject in    need thereof a therapeutically effective amount of the complex of    embodiment 56 or embodiment 57, thereby treating the cancer or    inhibiting the development of the tumor neovasculature.-   62. The method of embodiment 61, wherein the complex is administered    systemically or locally.-   63. The method of embodiment 61 or embodiment 62, further comprising    administering to the subject a therapeutically effective amount of    peripheral blood mononuclear cells (PBMCs).

1. A recombinant protein comprising a double stranded RNA (dsRNA)binding domain and a target-binding moiety.
 2. The recombinant proteinof claim 1, wherein said dsRNA binding domain and said target-bindingmoiety are connected by a spacer peptide.
 3. (canceled)
 4. Therecombinant protein of claim 1, wherein said dsRNA binding domaincomprises one or more double-stranded RNA-binding motif (dsRBM), i.e. analpha-beta-beta-beta-alpha fold.
 5. The recombinant protein of claim 4,wherein said one or more dsRBM is selected from a dsRBM of dsRNAdependent protein kinase (PKR), TRBP, PACT, Staufen, NFAR1, NFARZ, SPNR,RHA or NREBP.
 6. The recombinant protein of claim 5, wherein said dsRNAbinding domain comprises two dsRBMs of a PKR, optionally connected by aflexible linker.
 7. The recombinant protein of claim 6, wherein saiddsRNA binding domain is selected from the dsRNA binding domain of humanPKR and said two dsRBMs are connected by a flexible linker; or the fulllength human PKR.
 8. The recombinant protein of claim 7, wherein saiddsRNA binding domain comprises amino acid residues 1-197 of human PKR.9. The recombinant protein any one of claim 1, wherein saidtarget-binding moiety comprises (i) a ligand to a cell surface receptor;(ii) an antibody, such as a humanized antibody; a human antibody; afunctional fragment of an antibody; a single-domain antibody, such as aNanobody; a recombinant antibody; and a single chain variable fragment(scFv); (ii) an antibody mimetic, such as an affibody molecule; anaffilin; an affimer; an affitin; an alphabody; an anticalin; an avimer;a DARPin; a fynomer; a Kunitz domain peptide; and a monobody; or (iii)an aptamer.
 10. The recombinant protein of claim 9, wherein saidtarget-binding moiety binds a tumor-associated antigen.
 11. Therecombinant protein of claim 10, wherein said tumor-associated antigenis selected from epidermal growth factor receptor (EGFR), humanepidermal growth factor receptor 2 (HER2), prostate surface membraneantigen (PSMA), fibroblast growth factor receptor (FGFR), colonystimulating factor 1 receptor (CSF-1R), platelet-derived growth factorreceptors (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) andMET.
 12. The recombinant protein of claim 11, wherein saidtarget-binding moiety is an EGFR ligand, such as an EGF or the peptideGE11 of the sequence YHWYGYTPQNVI; an anti-EGFR antibody, such as ananti-EGFR scFv or a humanized or human anti-EGFR antibody; or ananti-EGFR affibody.
 13. The recombinant protein of claim 12, whereinsaid EGF is human EGF and said EGFR is human EGFR. 14-25. (canceled) 26.A complex comprising the recombinant protein of claim 1 and dsRNA. 27.The complex of claim 26, wherein said dsRNA is dsRNA comprising apolyinosinic acid strand and a polycytidylic acid strand (poly IC).28-31. (canceled)
 32. A nucleic acid molecule comprising a nucleic acidsequence encoding the recombinant protein of claim
 1. 33. The nucleicacid molecule of claim 32, wherein said sequence is optimized forexpression in a bacterial or plant host cell, preferably a plant hostcell.
 34. (canceled)
 35. A method for manufacturing a recombinantprotein comprising a dsRNA binding domain and a target-binding moiety,comprising expressing the nucleic acid molecule of claim 32 in abacterial or plant cell and extracting said recombinant protein fromsaid cells. 36-37. (canceled)
 38. The method of any one of claim 35,comprising expressing the nucleic acid molecule in a plant cell, such asa tobacco or carrot cell, either in suspension or in a whole plant. 39.A method for treatment of cancer characterized by expression of atumor-associated antigen, said method comprising systemicallyadministering to a patient in need the complex of claim
 26. 40-63.(canceled)